Synthetic peptide constructs for the diagnosis and treatment of periodontitis associated with porphyromonas gingivalis

ABSTRACT

The present invention relates to an oral composition and an immunogenic composition for the suppression of the pathogenic effects of the intra-oral bacterium  Porphyromonas gingivalis  associated with periodontal disease.

FIELD OF THE INVENTION

[0001] This invention relates to an oral composition and an immunogeniccomposition for the suppression of the pathogenic effects of theintra-oral bacterium Porphyromonas gingivalis associated withperiodontal disease. It also relates to diagnostic tests for thepresence of Porphyromonas gingivalis in subgingival plaque samples andspecific antibodies against P. gingivalis antigens in sera. Thecompositions comprise synthetic peptide constructs corresponding tostructurally and functionally significant areas of the PrtR-PrtKproteinase-adhesin complex of Porphyromonas gingivalis. Also disclosedare methods for preparing the synthetic peptide constructs. Thesynthetic peptide constructs are useful as immunogens in raising animmune response against P. gingivalis and can be used to generateprotein-specific and peptide-specific antisera useful for passiveimmunization and as reagents for diagnostic assays.

BACKGROUND OF THE INVENTION

[0002] Periodontal diseases are bacterial-associated inflammatorydiseases of the supporting tissues of the teeth and range from therelatively mild form of gingivitis, the non-specific, reversibleinflammation of gingival tissue to the more aggressive forms ofperiodontitis which are characterised by the destruction of the tooth'ssupporting structures. Periodontitis is associated with a subgingivalinfection of a consortium of specific Gram-negative bacteria that leadsto the destruction of the periodontium and is a major public healthproblem. One bacterium that has attracted considerable interest isPorphyromonas gingivalis as the recovery of this microorganism fromadult periodontitis lesions can be up to 50% of the subgingivalanaerobically cultivable flora, whereas P. gingivalis is rarelyrecovered, and then in low numbers from healthy sites. A proportionalincrease in the level of P. gingivalis in subgingival plaque has beenassociated with an increased severity of periodontitis and eradicationof the microorganism from the cultivable subgingival microbialpopulation is accompanied by resolution of the disease. The progressionof periodontitis lesions in non-human primates has been demonstratedwith the subgingival implantation of P. gingivalis. These findings inboth animals and humans suggest a major role for P. gingivalis in thedevelopment of adult periodontitis.

[0003]P. gingivalis is a black-pigmented, anaerobic, proteolyticGram-negative rod that obtains energy from the metabolism of specificamino acids. The microorganism has an absolute growth requirement foriron, preferentially in the form of heme or its Fe(III) oxidationproduct hemin and when grown under conditions of excess hemin is highlyvirulent in experimental animals. A number of virulence factors havebeen implicated in the pathogenicity of P. gingivalis including thecapsule, adhesins, cytotoxins and extracellular hydrolytic enzymes. Inorder to develop an efficacious and safe vaccine to prevent P.gingivalis colonisation it is necessary to identify effective antigensthat are involoved in virulence that have utility as immunogens togenerate neutralising antibodies.

[0004] We have purified and characterised a 300 kDa multiprotein complexof cysteine proteinases and adhesins which is a major virulence factorfor P. gingivalis. This complex was biochemically characterised anddisclosed in International Patent Application No. PCT/AU96/00673, thedisclosure of which is incorporated herein by reference. The complexconsists of a 160 kDa Arg-specific proteinase with C-terminal adhesindomains (designated PrtR) associated with a 163 kDa Lys-specificproteinase also with C-terminal adhesin domains (designated PrtK). TheC-terminal adhesin domains of the PrtR and PrtK have homology with allhaemagglutinin from P. gingivalis designated HagA. The gene encodingHagA has been disclosed in the international patent WO96/17936, thedisclosure of which is incorporated herein by reference.

SUMMARY OF THE INVENTION

[0005] The present inventors have identified a number of structurallyand functionally significant sequences from the 300 kDa multiproteincomplex of cysteine proteinases and adhesins which is a major virulencefactor for P. gingivalis. These sequences are set out in Table 1. TABLE1 Amino acid sequences of the PrtR-PrtK proteinase-adhesin complex offunctional significance. Sequence (single letter code) DesignationProteinase Active Site PrtR45 (426-446) FNGGISLANYTGHGSETAWGT PAS1 (R45)PrtK48 (432-453) LNTGVSFANYTAHGSETAWADP PAS1 (K48) PrtR45 (467-490)FDVACVNGDFLFSMPCFAEALMRA PAS2 (R45) PrtK48 (473-496)IGNCCITAQFDYVQPCFGEVITRV PAS2 (K48) Adhesin Binding Motif PrtR45(660-689) GEPNPYQPVSNLTATTQGQKVTLKWDAPSTK ABM1 (R45) PrtR44 (919-949)EGSNEFAPVQNLTGSAVGQKVTLKWDAPNGT ABM1 (R44) PrtR17 (1375-1405)VNSTQFNPVKNLKAQPDGGDVVLKWEAPSAK ABM1 (R17) PrtK48 (681-711)GEPSPYQPVSNLTATTQGQKVTLKWEAPSAK ABM1 (K48) PrtK39 (940-970)EGSNEFAPVQNLTGSSVGQKVTLKWDAPNGT ABM1 (K39) PrtK44 (1393-1425)VNSTQFNPVQNLTAEQAPNSMDAILKWNAPASK ABM1 (K44) HagA (1837-1863)QFNPVQNLTGSAVGQKVTLKWDAPNGT ABM1 (HagA1) HagA (1381-1407)QFNPVQNLTGSAVGQKVTLKWDAPNGT ABM1 (HagA2) HagA (925-951)QFNPVQNLTGSAVGQKVTLKWDAPNGT ABM1 (HagA3) HagA (474-499)FAHVQNLTGSAVGQKVTLKWDAPNGT ABM1 (HaGA4) HagA (202-227)FAPVQNLQWSVSGQTVTLTWQAPASD ABM1 (HagA5) HagA (2293-2321)QFNPVQNLTAEQAPNSMDAILKWNAPASK ABM1 (HagA6) PrtR44 (865-893)DYTYTVYRDGTKIKEGLTATTFEEDGVAT ABM2 (R44) PrtR17 (1322-1350)DYTYTVYRDGTKIKEGLTETTFEEDGVAT ABM2 (R17) PrtR27 (1580-1608)SYTYTVYRDGTKIKEGLTETTYRDAGMSA ABM2 (R27) PrtK39 (886-914)SYTYTVYRDGTKIKEGLTATTFEEDGVAA ABM2 (K39) PrtK44 (1340-1368)DYTYTVYRDGTKIKEGLTETTFEEDGVAT ABM2 (K44A) PrtK44 (1606-1634)SYTYTIYRNNTQIASGVTETTYRDPDLAT ABM2 (K44B) HagA (2236-2264)DYTYTVYRDGTKIKEGLTETTFEEDGVAT ABM2 (HagA1) HagA (1780-1808)DYTYTVYRDGTKIKEGLTETTFEEDGVAT ABM2 (HagA2) HagA (1324-1352)DYTYTVYRDGTKIKEGLTETTFEEDGVAT ABM2 (HagA3) HagA (868-896)DYTYTVYRDGTKIKEGLTETTFEEDGVAT ABM2 (HagA4) HagA (415-443)DYTYTVYRDNVVIAQNLAATTFNQENVAP ABM2 (HagA5) HagA (2502-2530)SYTYTIYRNNTQIASGVTETTYRDPDLAT ABM2 (HagA6) PrtR44 (946-971)PNGTPNPNPNPNPNPNPGTTTLSESF ABM3 (R44) PrtK39 (967-989)PNGTPNPNPNPNPNPGTTLSESF ABM3 (K39) HagA (1860-1881)PNGTPNPNPNPNPGTTTLSESF ABM3 (HagA1) HagA (1404-1425)PNGTPNPNPNPNPGTTTLSESF ABM3 (HagA2) HagA (948-969)PNGTPNPNPNPNPGTTTLSESF ABM3 (HagA3) HagA (496-513) PNGTPNPNPGTTTLSESFABM3 (HagA4) PrtR17 (1278-1297) WIERTVDLPAGTKYVAFRHY ABM4 (R17) PrtR44(1028-1043) WRQKTVDLPAGTKYVAFRHF ABM4 (R44) PrtK44 (1296-1315)WIERTVDLPAGTKYVAFRHY ABM4 (K44A) PrtK44 (1565-1584) WRQKTVDLPAGTKYVAFRHFABM4 (K44B) PrtK39 (1116-1135) WYQKTVQLPAGTKYVAFRHF ABM4 (K39) HagA(2191-2211) WIERTVDLPAGTKYVAFRHY ABM4 (HagA1) HagA (1736-1755)WIERTVDLPAGTKYVAFRHY ABM4 (HagA2) HagA (1280-1299) WIERTVDLPAGTKYVAFRHYABM4 (HagA3) HagA (824-843) WIERTVDLPAGTKYVAFRHY ABM4 (HagA4) HagA(2012-2031) WYQKTVQLPAGTKYVAFRHF ABM4 (HagA5) HagA (1556-1575)WYQKTVQLPAGTKYVAFRHF ABM4 (HagA6) HagA (2461-2480) WYQKTVQLPAGTKYVAFRHFABM4 (HagA7) HagA (1100-1119) WYQKTVQLPAGTKYVAFRHF ABM4 (HagA8) HagA(644-663) WYQKTVQLPAGTKYVAFRHF ABM4 (HagA9) HagA (372-392)ERTIDLSAYAGQQVYLAFRHF ABM4 (HagA10) PrtR15 (1154-1169) PAEWTTIDADGDGQGWABM5 (R15) PrtR44 (976-991) PASWKTIDADGDGHGW ABM5 (R44) PrtK15(1172-1187) PAEWTTIDADGDGQGW ABM5 (K15) PrtK39 (994-1009)PASWKTIDADGDGHGW ABM5 (K39) PrtK44 (1439-1454) PASWKTIDADGDGNNW ABM5(K44) HagA (2068-2083) PAEWTTIDADGDGQGW ABM5 (HagA1) HagA (1612-1627)PAEWTTIDADGDGQGW ABM5 (HagA2) HagA (1156-1171) PAEWTTIDADGDGQGW ABM5(HagA3) HagA (700-715) PAEWTTIDADGDGQGW ABM5 (HagA4) HagA (1430-1445)PASWKTIDADGDGNNW ABM5 (HagA5) HagA (974-989) PASWKTIDADGDGNNW ABM5(HagA6) HagA (1886-1901) PASWKTIDADGDGNNW ABM5 (HagA7) HagA (518-533)PASWKTIDADGDGNNW ABM5 (HagA8) HagA (2335-2350) PSSWKTIDADGDGNNW ABM5(HagA9) HagA (243-258) PNGWTMIDADGDGHNW ABM5 (HagA10) PrtR44 (919-938)EGSNEFAPVQNLTGSAVGQK ABM6 (R44) PrtR45 (659-678) GEPNPYQPVSNLTATTQGQKABM6 (R45) PrtK39 (940-959) EGSNEFAPVQNLTGSSVGQK ABM6 (K39) PrtK48(681-700) GEPSPYQPVSNLTATTQGQK ABM6 (K48) PrtK44 (1394-1412)NSTQFNPVQNLTAEQAPNS ABM6 (K44) HagA (469-488) EGSNEFAHVQNLTGSAVGQK ABM6(HagA1) HagA (1834-1852) DPVQFNPVQNLTGSAVGQK ABM6 (HagA2) HagA(1378-1396) DPVQFNPVQNLTGSAVGQK ABM6 (HagA3) HagA (922-940)DPVQFNPVQNLTGSAVGQK ABM6 (HagA4) HagA (197-216) EGGNEFAPVQNLQWSVSGQTABM6 (HagA5) HagA (2290-2308) NPTQFNPVQNLTAEQAPNS ABM6 (HagA6) PrtR44(894-918) GNHEYCVEVKYTAGVSPKVCKDVTV ABM7 (R44) PrtR17 (1351-1375)GNHEYCVEVKYTAGVSPKKCVNVTV ABM7 (R17) PrtR27 (1610-1630)SHEYCVEVKYTAGVSPKVCVD ABM7 (R27) PrtK39 (915-939)GNHEYCVEVKYTAGVSPKVCKDVTV ABM7 (K39) PrtK44 (1369-1393)GNHEYCVEVKYTAGVSPKKCVNVTV ABM7 (K44) HagA (2265-2289)GNHEYCVEVKYTAGVSPKVCVNVTI ABM7 (Hag1) HagA (444-468)GQYNYCVEVKYTAGVSPKVCKDVTV ABM7 (Hag2) HagA (1809-1833)GNHEYCVEVKYTAGVSPEVCVNVTV ABM7 (Hag3) HagA (1353-1377)GNHEYCVEVKYTAGVSPEVCVNVTV ABM7 (Hag4) HagA (897-921)GNHEYCVEVKYTAGVSPEVCVNVTV ABM7 (Hag5)

[0006] Accordingly in a first aspect the present invention consists in acomposition for use in raising an immune response against Porphyromonasgingivalis, the composition including a suitable adjuvant and/oracceptable carrier or excipient and at least one peptide selected fromthe group consisting of:— FNGGISLANYTGHGSETAWGT; LNTGVSFANYTAHGSETAWADP;FDVACVANGDFLFSMPCFAEALMRA; IGNCCITAQFDYVQPCFGEVITRVGEPNPYQPVSNLTATTQGQKVTLKWDAPSTK EGSNEFAPVQNLTGSAVGQKVTLKWDAPNGT;VNSTQFNPVKNLKAQPDGGDVVLKWEAPSAK; GEPSPYQPVSNLTATTQGQKVTLKWEAPSAK;EGSNEFAPVQNLTGSSVGQKVTLKWDAPNGT; VNSTQFNPVQNLTAEQAPNSMDAILKWNAPASK;QFNPVQNLTGSAVGQKVTLKWDAPNGT; FAHVQNLTGSAVGQKVTLKWDAPNGT;FAPVQNLQWSVSGQTVTLTWQAPASD; QFNPVQNLTAEQAPNSMDAILKWNAPASK;DYTYTVYRDGTKIKEGLTATTFEEDGVAT; DYTYTVYRDGTKIKEGLTETTFEEDGVAT;SYTYTVYRDGTKIKEGLTETTYRDAGMSA; SYTYTVYRDGTKIKEGLTATTFEEDGVAA;DYTYTVYRDGTKIKEGLTETTFEEDGVAT; SYTYTIYRNNTQIASGVTETTYRDPDLAT;DYTYTVYRDNVVIAQNLAATTFNQENVAP; SYTYTIYRNNTQIASGVTETTYRDPDLAT;PNGTPNPNPNPNPNPNPGTTTLSESF; PNGTPNPNPNPNPNPGTTLSESF;PNGTPNPNPNPNPGTTTLSESF; PNGTPNPNPGTTTLSESF; WIERTVDLPAGTKYVAFRHY;WRQKTVDLPAGTKYVAFRHF; WYQKTVQLPAGTKYVAFRHF; ERTIDLSAYAGQQVYLAFRHF;PAEWTTIDADGDGQGW; PASWKTIDADGDGHGW; PASWKTIDADGDGNNW; PSSWKTIDADGDGNNW;PNGWTMIDADGDGHNW; EGSNEFAPVQNLTGSAVGQK; GEPNPYQPVSNLTATTQGQK;EGSNEFAPVQNLTGSSVGQK; GEPSPYQPVSNLTATTQGQK; NSTQFNPVQNLTAEQAPNS;EGSNEFAHVQNLTGSAVGQK; DPVQFNPVQNLTGSAVGQK; EGGNEFAPVQNLQWSVSGQT;NPTQFNPVQNLTAEQAPNS; GNHEYCVEVKYTAGVSPKVCKDVTV;GNHEYCVEVKYTAGVSPKKCVNVTV; SHEYCVEVKYTAGVSPKVCVD;GNHEYCVEVKYTAGVSPKKCVNVTV; GNHEYCVEVKYTAGVSPKVCVNVTI;GQYNYCVEVKYTAGVSPKVCKDVTV; and GNHEYCVEVKYTAGVSPEVCVNVTV.

[0007] In a preferred embodiment of the first aspect of the presentinvention, the composition includes at least one peptide selected fromthe group consisting of:— FNGGISLANYTGHGSETAWGT; LNTGVSFANYTAHGSETAWADP;PYQPVSNLTATTQGQKVTLKWDAPSTK; SYTYTVYRDGTKIKEGLTATTFEEDGVAA;VTLKWDAPNGTPNPNPNPNPNPNPGTTTLSESF; WIERTVDLPAGTKYVAFRHY;PAEWTTIDADGDGQGW; and EGSNEFAPVQNLTGSAVGQK.

[0008] Where the composition includes more than one peptide the peptidesmay be present in the composition as individual peptides or inmultimeric forms. Where multimeric forms are used the multimer maycomprise multiple copies of the same peptide, however, it is preferredthat the multimer includes differing peptides.

[0009] Peptides (PAS1 and PAS2) of Table 1 represent sequences of theArg-specific (PrtR45) and Lys-specific (PrtK48) cysteine proteinaseswhich form the active site containing the Cys-His catalytic dyad.

[0010] The remaining peptides (ABM peptides) represent adhesin bindingmotifs of the PrtR-PrtK protein-adhesin complex and HagA and togetherwith the proteinase active site sequences, have proven to be effectiveas synthetic peptide vaccines.

[0011] In a second aspect the present invention consists in a peptide,the peptide being selected from the group consisting of:—FNGGISLANYTGHGSETAWGT; LNTGVSFANYTAHGSETAWADP; FDVACVNGDFLFSMPCFAEALMRA;IGNCCITAQFDYVQPCFGEVITRV; GEPNPYQPVSNLTATTQGQKVTLKWDAPSTK;EGSNEFAPVQNLTGSAVGQKVTLKWDAPNGT; VNSTQFNPVKNLKAQPDGGDVVLKWEAPSAK;GEPSPYQPVSNLTATTQGQKVTLKWEAPSAK; EGSNEFAPVQNLTGSSVGQKVTLKWDAPNGT;VNSTQFNPVQNLTAEQAPNSMDAILKWNAPASK; QFNPVQNLTGSAVGQKVTLKWDAPNGT;FAHVQNLTGSAVGQKVTLKWDAPNGT; FAPVQNLQWSVSGQTVTLTWQAPASD;QFNPVQNLTAEQAPNSMDAILKWNAPASK; DYTYTVYRDGTKIKEGLTATTFEEDGVAT;DYTYTVYRDGTKIKEGLTETTFEEDGVAT; SYTYTVYRDGTKIKEGLTETTYRDAGMSA;SYTYTVYRDGTKIKEGLTATTFEEDGVAA; DYTYTVYRDGTKIKEGLTETTFEEDGVAT;SYTYTIYRNNTQIASGVTETTYRDPDLAT; DYTYTVYRDNVVIAQNLAATTFNQENVAP;SYTYTIYRNNTQIASGVTETTYRDPDLAT; PNGTPNPNPNPNPNPNPGTTTLSESF;PNGTPNPNPNPNPNPGTTLSESF; PNGTPNPNPNPNPGTTTLSESF; PNGTPNPNPGTTTLSESF;WIERTVDLPAGTKYVAFRHY; WRQKTVDLPAGTKYVAFRHF; WYQKTVQLPAGTKYVAFRHF;ERTIDLSAYAGQQVYLAFRHF; PAEWTTIDADGDGQGW; PASWKTIDADGDGHGW;PASWKTIDADGDGNNW; PSSWKTIDADGDGNNW; PNGWTMIDADGDGHNW;EGSNEFAPVQNLTGSAVGQK; GEPNPYQPVSNLTATTQGQK; EGSNEFAPVQNLTGSSVGQK;GEPSPYQPVSNLTATTQGQK; NSTQFNPVQNLTAEQAPNS; EGSNEFAHVQNLTGSAVGQK;DPVQFNPVQNLTGSAVGQK; EGGNEFAPVQNLQWSVSGQT; NPTQFNPVQNLTAEQAPNS;GNHEYCVEVKYTAGVSPKVCKDVTV; GNHEYCVEVKYTAGVSPKKCVNVTV;SHEYCVEVKYTAGVSPKVCVD; GNHEYCVEVKYTAGVSPKKCVNVTV;GNHEYCVEVKYTAGVSPKVCVNVTI; GQYNYCVEVKYTAGVSPKVCKDVTV; andGNHEYCVEVKYTAGVSPEVCVNVTV.

[0012] In a preferred embodiment of the second aspect of the presentinvention, the peptide is selected from the group consisting of:—FNGGISLANYTGHGSETAWGT; LNTGVSFANYTAHGSETAWADP;PYQPVSNLTATTQGQKVTLKWDAPSTK; SYTYTVYRDGTKIKEGLTATTFEEDGVAA;VTLKWDAPNGTPNPNPNPNPNPNPGTTTLSESF; WIERTVDLPAGTKYVAFRHY;PAEWTTIDADGDGQGW; and EGSNEFAPVQNLTGSAVGQK.

[0013] As will be readily apparent to persons skilled in this area thesepeptides may be used as antigens in diagnostic tests or as immunogens informulations.

[0014] In a third aspect the present invention consists in an antibodypreparation comprising antibodies specifically directed against thecomposition of the first aspect of the invention or the peptides of thesecond aspect of the invention. The antibodies may be either polyclonalor monoclonal antibodies.

[0015] In a fourth aspect the present invention consists in a method oftreating a subject suffering from Porphyromonas gingivalis infection,the method comprising administering to the subject an effective amountof the antibody preparation of the third aspect.

[0016] In a preferred embodiment the antibody preparation isadministered as a mouth wash or as a dentifrice.

[0017] In a fifth aspect the present invention consists in a method ofreducing the prospect of P. gingivalis infection in an individual and/orseverity of disease, the method comprising administering to theindividual an amount of the composition of the first aspect effective toinduce an immune response in the individual directed against P.gingivalis.

[0018] Peptides can be synthesized using one of the several methods ofpeptide synthesis known in the art including standard solid phasepeptide synthesis using t-butyloxycarbonyl amino acids (Mitchell et al.,1978, J. Org. Chem. 43:2845-2852) using 9-fluorenylmethyloxycarbonyl(Fmoc) amino acids on a polyamide support (Druland et al., 1986, J.Chem. Soc.Perkin Trans. 1 125-137) by pepscan synthesis (Geysen et al.,1987, J. Immunol Methods 03:259; 1984, Proc. Natl. Acad. Sci. USA,81:3998) or by standard liquid phase synthesis.

[0019] A variety of methods for the synthesis ofmultivalent/multipeptide high molecular weight peptide molecules can beused to synthesize the peptide antigens. This will be achieved usingknown in the art and novel ligation strategies.

[0020] Preparation of Synthetic Peptides

[0021] Peptides from Table 1 can be synthesized in such away as tocontain two ligands, which can be the same or different, which may ormay not be the complementary ligand. These bi-modal peptides canincorporate any

[0022] As will be readily apparent to persons skilled in this area thesepeptides may be used as antigens in diagnostic tests or as immunogens informulations.

[0023] In a third aspect the present invention consists in an antibodypreparation comprising antibodies specifically directed against thecomposition of the first aspect of the invention or the peptides of thesecond aspect of the invention. The antibodies may be either polyclonalor monoclonal antibodies.

[0024] In a fourth aspect the present invention consists in a method oftreating a subject suffering from Porphyromonas gingivalis infection,the method comprising administering to the subject an effective amountof the antibody preparation of the third aspect.

[0025] In a preferred embodiment the antibody preparation isadministered as a mouth wash or as a dentifrice.

[0026] In a fifth aspect the present invention consists in a method oftreating a subject suffering from Porphyromonas gingivalis infection themethod comprising administering to the subject an effective amount of acomposition of the first aspect of the invention or a peptide of thesecond aspect of the invention.

[0027] In a preferred embodiment the composition or peptide isadministered as a mouth wash or as a dentifrice.

[0028] In a sixth aspect the present invention consists in a method ofreducing the prospect of P. gingivalis infection in an individual and/orseverity of disease, the method comprising administering to theindividual an amount of the composition of the first aspect effective toinduce an immune response in the individual directed against P.Gingivalis.

[0029] Throughout this specification, unless the context requiresotherwise, the word “comprise”, or variations such as “comprises” or“comprising”, will be understood to imply the inclusion of a statedelement or integer or group of elements or integers but not theexclusion of any other element or integer or croup of elements orintegers.

[0030] Peptides can be synthesized using one of the several methods ofpeptide synthesis known in the art including standard solid phasepeptide synthesis using t-butyloxycarbonyl amino acids (Mitchell et al.,1978. J. Ore. Chem. 43:2845-2852) using 9-fluorenylmethyloxycarbonyl(Fmoc) amino acids on a polyamide support (Druland et al., 1986. J.Chem, Soc.Perkin Trans. 1 125-137) by pepscan synthesis (Geysen et al.,1987. J. Immunol Methods 03:259; 1984, Proc. Natl. Acad. Sci. USA,81:3998) or by standard liquid phase synthesis.

[0031] A variety of methods for the synthesis ofmultivalent/multipeptide high molecular weight peptide molecules can beused to synthesize the peptide antigens. This will be achieved usingknown in the art and novel ligation strategies.

[0032] Preparation of Synthetic Peptides

[0033] Peptides from Table 1 can be synthesized in such away as tocontain two ligands, which can be the same or different, which may ormay not be the complementary ligand. These bi-modal peptides canincorporate any ligand thus linkages such as thioether, thioester,hydrazone, oxime, thiazolidine can be utilised for the synthesis ofmultipeptide constructs Shao and Tam., 1995, J. Am. Chem. Soc. 117,3893-3899, Rose, et al 1996, Bioconjugate Chem. 7(5):552-556. Rose. K.,1994, J. Am. Chem. Soc. 116:30-33, Canne., et al 1995, J. Am. Chem. Soc.117:2998-3007, Lu., et al. 1991, Mol. Immunol 28(6):623-630, Liu andTam., 1994, Proc. Natl. Acad. Sci. 91.:6584-6588. A novel ligatingstrategy is to use the known reaction between thioanisole and acryloylpeptides (O'Brien-Simpson et al., 1997. J. Am. Chem. Soc. 119 (6) whichresults in the para substitution of thioanisole by the double bond inacidic conditions. By synthesising and mixing acryloyl-peptides andphenylthio acetyl peptides and exposing them to acidic conditionsligation can proceed by Friedal-Craft alkylation. Ligation can beaccomplished between peptides and on to an oligolysine supportderivatised with one of the ligands. Conditions for ligation can consistof; Friedal-Craft reaction conditions which are known in the art andknown peptide cleavage conditions.

[0034] The introduction of ligand groups to form bi-modal peptides canbe achieved by coupling a ligand on to free amino groups, which is knownin the art at the N- or C-terminus of a peptide or within the peptidesequence. This can be achieved by coupling eg. Fmoc(Fmoc) 2,3 diaminopropionic acid or Fmoc Lys (Fmoc)-OH or orthogonally protected lysineresidues such as Fmoc Lys (Mtt)-OH using standard peptide couplingprotocols on to the N-terminus or introduced at the C-terminus or withinthe peptide sequence. After deprotection, ligand groups can be coupledon to the amino groups and by selective deprotection of eg. Fmoc Lys(Mtt) different ligands can be coupled on to a single peptide. At anypoint in the synthesis spacer moieties can be introduced between thepeptide and the ligands and/or between the ligands, which may be used toreduce steric hindrance in the ligation reaction. FIG. 1 shows thesynthesis protocol.

[0035] Peptide ligation can be achieved in solution or on the solidphase. The incorporation of different ligands and selective protectionof one ligand can allow the synthesis of multivalent, multipeptideconstructs, where by, peptides are ligated sequentially. This strategyhas the advantage that the orientation and order of peptides ligated isknown and can be controlled. Protecting groups for ligands can be forexample Fmoc, allyloxycarbonyl (Aloc) or nitrocinnamyloxycarbonyl (Noc)which are stable to standard cleavage conditions but are easily removedunder basic conditions or catalytic allyl transfer. FIG. 2 shows theligation scheme for the synthesis of multivalent peptide constructsusing bi-modal peptides. The protocol can be adapted for a variety ofligation chemistries by simply altering the ligands which are coupled tothe peptide to form the bi-modal peptide.

[0036] The step wise addition of each peptide can be achieved on thesolid phase. This can be achieved by synthesising a peptide on to thesolid support via a base labile handle eg. 4-hydroxymethyl benzoic acid.This can allow full side chain deprotection of the peptide with thepeptide remaining attached to the solid support. This would allowligation to still be carried out in aqueous solvents similar to thoseused for solution phase ligation except that separation of the ligandproduct from unreacted bi-modal peptide can be achieved by simplywashing the solid support. The reaction can be monitored by ninhydrin ortrinitrobenzene sulphonic acid tests, where by lysine residues withinthe bi-modal peptide would need to be protected eg. with(4.4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl (Dde) which is stable toacid cleavage but can be removed with hydrazine. FIG. 3 shows theligation strategy for the solid phase.

[0037] Bi-modal peptides can be synthesized so that ligands are at theN- and C-terminus. This would allow the preparation of cyclic peptidesand the formation of di-peptide constructs where by peptides can runparallel or anti parallel to each other by either coupling N-to N- andC-to C-termini or N-to C-termini together respectively (FIG. 4).

[0038] Another technique for the synthesis of multivalent peptideconstructs is to ligate peptides on to an oligolysine support (Rose, etal 1996, Bioconjugate Chem. 7(5):552-556, Canne., et al 1995, J. Am.Chem. Soc. 117:2998-3007 and Lu., et al, 1991, Mol. Immunol28(6):623-630). By incorporating a number of different ligands and orprotected ligands on to the lysine support, peptides can be ligated to aparticular position on the support. Ligation chemistries such as oximeor hydrazone with haloacylation and Friedal-Craft alkylation can be usedsequentially without the need for ligand protection. Ligand protectioncan be used to increase the number of different peptides incorporated onto the lysine support. FIG. 5 demonstrates the synthesis protocol.

[0039] Another method known in the art is the synthesis of acryloylpeptides and their polymerisation with acrylamide (O'Brien-Simpson etal., 1997, J. Am. Chem. Soc. 119 (6)) or acryloyl amino acids. Peptidesfrom the PrtR-PrtK protein complex listed in Table 1 can be acryloylatedand polymerised either singularly or in combination. Although thismethod allows the polymerisation of a number of peptides together theorder in which peptides are incorporated can not be controlled.

[0040] The final peptide construct may or may not contain all, sum orpart of the peptides listed in Table 1. Also the construct may or maynot contain promiscuous T-cell epitopes known in the art (Kaumaya et al1994. in Solid Phase Synthesis. Ed Epton, R) or a derived sequence fromstructural/binding motifs of MHC class II binding peptides (O'Sullivanet al., 1991, J. Immunol, 147:2663-2669, Hammer et al., 1993, Cell,74:197-203 and Alexander et al., 1994, Immunity, 1:751-761).Furthermore, lipid moieties such as palmitic acid or cholesterol can beincluded to enhance the immunogenic properties of the peptide construct.Enzymatic cleavable sequences known in the art (Duncan et al., ref) orderived sequences from cleavage motifs (Van Noort and van der Drift.,ref) can also be incorporated with the peptide construct.

[0041] The synthetic peptide antigens identified in Table 1 are ofparticular interest for diagnostics and neutralisation by passiveimmunity through oral compositions containing neutralising antibodiesand by vaccine development. The superiority of these synthetic peptideantigens to prior disclosed P. gingivalis antigens, is that thesesequences are homologous to structurally and functionally significantareas of the major P. gingivalis virulence factor the PrtR-PrtKproteinase-adhesin complex. The peptides represent sequences associatedwith the active site of the proteinases and binding domains of theadhesins making them ideal for the development of diagnostic andimmunoprophylactic products.

[0042] Antibodies against the antigens can be used in oral compositionssuch as toothpaste and mouthwash to neutralise the antigens and thusprevent disease. Antigen-specific antibodies can also be used for theearly detection of P. gingivalis in subgingival plaque samples by adiagnostic assay. A vaccine based on these antigens and suitableadjuvant delivered by nasal spray, orally or by injection to produce aspecific immune response against these antigens thereby reducingcolonisation and virulence of P. gingivalis and thereby preventingdisease. The peptide antigens of the present invention may be used asimmunogens in prophylactic and/or therapeutic vaccine formulations; oras an antigen in diagnostic immunoassays directed to detection of P.gingivalis infection by measuring an increase in serum titer of P.gingivalis—specific antibody. Also the synthetic peptides of the presentinvention may be used to generate antigen-specific antibody which may beuseful for passive immunization and as reagents for diagnostic assaysdirected to detecting the presence of P. gingivalis in clinicalspecimens such as subgingival plaque samples.

[0043] Unlike whole P. gingivalis cells or other previously preparedantigens, the synthetic peptide antigens described herein are safe andeffective antigens for the preparation of a vaccine for the preventionof P. gingivalis—associated periodontal disease.

BRIEF DESCRIPTION OF THE FIGURES

[0044]FIG. 1: Synthesis of Bi-modal Peptides Although a specific exampleis shown here any ligand can be introduced at the a or e amino groups oflysine. (a) acylation e.g. amino acid:HOBt:HBTU:DIPEA 1:1:1:1.5 indimethyl formamide (DMF). (b) Fmoc deprotection e.g. 20% piperidine inDMF. (c) Levulinic acid: diisopropyl carbodiimide (DIC) 2:1 indichloromethane (DCM). 1 h. (d) Mtt removal, 3×1% TFA in DCM. 3 mins.(e) Fmoc-Hydrazino benzoic acid : DIC 2:1, in DCM, 1 h. (f) Acidcleavage e.g. TFA: water 95:5.

[0045]FIG. 2: Synthesis of multivalent peptide constructs using bi-modalpeptides. (a) Ligation. 8 M urea and 0.1 M NaH₂PO₄ (pH range 3-4.7).Ligation can be monitored by reverse phase analytical HPLC and massspectrometry. (b) Deprotection, e.g. Aloc is removed bypalladium(0)-catalyzed allyl gropu transfer to a basic receptor. Theligation product can be purified by preparative HPLC and lypholised. (c)Ligation. Similar conditions as described in (a). Different ligationchemistries can be used by synthesising peptides with different ligandsand synthesising non-complementary ligands on to the same peptide,thereby avoiding proected ligands. The square symbol indicatesprotection, (L) ligand, (P) peptide.

[0046]FIG. 3: Synthesis of multivalent peptide constructs using bi-modalpeptides by solid phase. (a) Deprotection and ligation. The S-acetylprotecting group is removed by aqueous hydroxyamine 0.05 M, pH 7.3.After washing the first peptide can be ligated on to the SH group, 6 Maqueous guanidine hydrochloride and 0.05 M EDTA pH 6.4-6.5 adjusted by 1M Tris.HCl under nitrogen. Ligation buffer can contain organic solventssuch as acetonitrile. (b) Deprotection, the S-acetyl protecting groupcan be removed by aqueous hydroxyamine 0.05 M, pH 7.3. (c) Ligation, asdescribed in (a) although different ligation chemistries can be used bysynthesising peptides with different ligands and synthesisingnon-complementary ligands on to the same peptide, thereby avoidingproected ligands. The square symbol indicates protection, (L) ligand,(P) peptide, (B) base labile handle. 4-hydroxymethyl benzoic acid.

[0047]FIG. 4: Cyclization using bi-modal peptides. (a) Deprotection andcyclisation. Synthesis of bi-modal peptides which have complimentaryligands at their N- and C-termini allows the cyclisation of thesepeptides in aqueoous buffers. (i) Ligation. (ii) Deprotection andligaction. (iii) Cleavage of the cyclic peptide from the base labilehandle. Example: The peptides shown are from Table 1 and present theactive site peptides from prtR 45. (a) Ligation. 95% aqueous TFA.Ligation can be monitored by reverse phase analytical HPLC and massspectrometry. Ligation conditions can be varied to included scavangerscommonly used in peptide synthesis and different acidic conditions toenhance the Friedal-Craft alkylation. (b) Deprotection and ligation. TheS-acetyl protecting group can removed by aqueous hydroxyamine 0.05 M, pH7.3. Ligation, 6 M aqueous guanidine hydrochloride and 0.05 M EDTA pH6.4-6.5 adjusted by 1 M Tris.HCl under nitrogen. The ligation straegycan also be accomplished on the solid phase. By selecting which ligandto introduce at the N- and C-terminal parallel and anti-parallel cyclicpeptides can be synthesised.

[0048]FIG. 5: Synthesis of multivalent multiple antigenic peptides(MAPs) using alternate ligation chemistries. By using different ligationstrategies a vareity of peptides can be ligated onto a single multipleantigenic peptide. The example shown is of peptides listed in Table 1.(a) Ligation, 95% aqueous TFA. Ligation can be monitored by reversephase analytical HPLC and mass spectrometry. Deprotection, Aloc canremoved by palladium(0)-catalyzed allyl group transfer to a basicreceptor after purifaction the second peptide can be ligated on to theMAP, (c) 8 M urea and 0.1 M NaH₂PO₄ (pH range 3-4.7).

[0049]FIG. 6 Gel Filtration FPLC of pooled and concentrated fractionseluting from Q sepharose anion exchange FPLC. Anion exchange fractionseluting between 160-246 mM NaCl and representing the leading edge of themain peak of proteolytic/amidolytic activity were pooled, equilibratedin TC buffer pH 7.4 containing 50 mM NaCl, concentrated and applied toSuperose 12 HR 10/30 gel filtration column using the same buffer at aflow rate of 0.3 ml min⁻¹. Fractions (0.5 ml) were assayed forproteolytic/amidolytic activity using azocasein, BZ-L-Arg-pNA andz-L-Lys-pNA. Amidolytic activity of each 0.5 ml fraction with BZ-L-pNAis shown by the histogram.

[0050]FIG. 7 SDS-PAGE (bolied/reduced conditions) of the anion exchange(Mono Q) peak eluting at 200 mM NaCl and containing only Arg-specificactivity. Lane 1, Pharmacia low molecular mass standards; lane 2,Purified 50 kDa Arg-specific proteinase, PrtRII50.

[0051]FIG. 8 An alignment of the deduced amino acid sequences ofPrtRII50, PrtR45 Arg-specific proteinase and PrtK48 Lys-specificproteinase with optimised similarity. The amino acyl residues ofPrtRII50 are numbered from the N-terminal residue of the matureprotein. * indicates an identical residue

[0052]FIG. 10 Competitive Binding Assay demonstrating binding of theTLCK-inactivated PrtR-PrtK proteinase-adhesion complex to the syntheticpeptide corresponding to the putative adhesin binding motif (ABM).-^()-^()-ABM synthetic peptide.

[0053] PYQPVSNLTATTQGQKVTLKWDAPSTK. -^(▪)-^(▪)-Control peptide,

[0054] FNGGISLANYTGHGSETAWGT corresponding to residues 428-448 ofPrtR45.

[0055] -^(▴)-^(▴)casein. See Materials and Methods for details.

[0056]FIG. 11. Average lesion size of mice challenged with Porphyromonasgingivalis in a mouse abcsess model. BALB/c mice (6 per group) wereinoculated (s.c.) with 50 kg of antigen emulsified in CFA and IFA forthe primary and secondary inoculations and then challenged (s.c.) with8×10⁹ cells of P. gingivalis strain 33277.

[0057] ABM1(R45)-DT, (□); ABM2(K39)-KT, (◯); ABM3(R44)-DT, (*);

[0058] ABM4(R17)-DT, (): ABM5(R15)-DT, (♦); ABM6(K39)-DT, (⋄)

[0059] PAS1(R45)-DT, (▴); PAS1(K48)-DT, (^(▪)); Control peptide-DT,(-⋄-);

[0060] formalin killed P. gingivalis strain 33277, (+); DT, (- -Δ- - );adjuvant, (^(×)).

[0061] For clarity error bars are not shown.

DETAILED DESCRIPTION OF THE INVENTION

[0062] This invention relates to an oral composition and an immunogeniccomposition for the suppression of the pathogenic effects of theintra-oral bacterium Porphyromonas gingivalis associated withperiodontal disease. It also relates to diagnostic tests for thepresence of Porphyromonas gingivalis in subgingival plaque samples andspecific anti-P. gingivalis antibodies in sera. The peptide antigens ofTable 1 can be synthesized individually or as multimetric ormultipeptide constructs.

[0063] The synthetic peptide antigens are used to generate polyclonal ormonoclonal antibodies using standard techniques. The animals used forantibody generation can be mice, rabbits, goats, chickens, sheep,horses, cows etc. When a high antibody titre against the antigens isdetected by immunoassay the animals are bled or eggs or milk arecollected and the serum prepared and/or antibody purified using standardtechniques or monoclonal antibodies produced by fusing spleen cells withmyeloma cells using standard techniques. The antibody (immunoglobulinfraction) may be

[0064] This invention relates to an oral composition and an immunogeniccomposition for the suppression of the pathogenic effects of theintra-oral bacterium Porphyromonas gingivalis associated withperiodontal disease. It also relates to diagnostic tests for thepresence of Porphyromonas gingivalis in subgingival plaque samples andspecific anti-P. gingivalis antibodies in sera. The peptide antigens ofTable 1 can be synthesized individually or as multimetric ormultipeptide constructs.

[0065] The synthetic peptide antigens are used to generate polyclonal ormonoclonal antibodies using standard techniques. The animals used forantibody generation can be mice, rabbits, goats, chickens, sheep,horses, cows etc. When a high antibody titre against the antigens isdetected by immunoassay the animals are bled or eggs or milk arecollected and the serum prepared and/or antibody purified using standardtechniques or monoclonal antibodies produced by fusing spleen cells withmyeloma cells using standard techniques. The antibody (immunoglobulinfraction) may be separated from the culture or ascites fluid, serum,milk or egg by salting out, gel filtration, ion exchange and/or affinitychromatography, and the like, with salting out being preferred. In thesalting out method the antiserum or the milk is saturated with ammoniumsulphate to produce a precipitate, followed by dialyzing the precipitateagainst physiological saline to obtain the purified immunoglobulinfraction with the specific antibody. The preferred antibody is obtainedfrom the equine antiserum and the bovine antiserum and milk. In thisinvention the antibody contained in the antiserum and milk obtained byimmunising the animal with the antigens is blended into the oralcomposition. In this case the antiserum and milk as well as the antibodyseparated and purified from the antiserum and milk may be used. Each ofthese materials may be used alone or in combination of two or more.Antibodies can be used in oral compositions such as toothpaste andmouthwash to neutralise P. gingivalis and thus prevent disease. Theantibodies can also be used for the early detection of P. gingivalis insubgingival plaque samples by a chairside Enzyme Linked ImmunosorbentAssay (ELISA).

[0066] For oral compositions it is preferred that the amount of theabove antibodies administered is 0.0001-50 g/kg/day and that the contentof the above antibodies is 0.0002-10% by weight preferably 0.002-5% byweight of the composition. The oral composition of this invention whichcontains the above-mentioned serum or milk antibody may be prepared andused in various forms applicable to the mouth such as dentifriceincluding toothpastes, toothpowders and liquid dentifrices, mouthwashes,troches, chewing gums, dental pastes, gingival massage creams, gargletablets, dairy products and other foodstuffs. The oral compositionaccording to this invention may further include additional well knowningredients depending on the type and form of a particular oralcomposition.

[0067] In certain highly preferred forms of the invention the oralcomposition may be substantially liquid in character, such as amouthwash or rinse. In such a preparation the vehicle is typically awater-alcohol mixture desirably including a humectant as describedbelow. Generally, the weight ratio of water to alcohol is in the rangeof from about 1:1 to about 20:1. The total amount of water-alcoholmixture in this type of preparation is typically in the range of fromabout 70 to about 99.9% by weight of the preparation. The alcohol istypically ethanol or isopropanol. Ethanol is preferred.

[0068] The pH of such liquid and other preparations of the invention isgenerally in the range of from about 4.5 to about 9 and typically fromabout 5.5 to 8. The pH is preferably in the range of from about 6 toabout 8.0, preferably 7.4. The pH can be controlled with acid (e.g.citric acid or benzoic acid) or base (e.g. sodium hydroxide) or buffered(as with sodium citrate, benzoate, carbonate, or bicarbonate, disodiumhydrogen phosphate, sodium dihydrogen phosphate, etc).

[0069] Other desirable forms of this invention, the oral composition maybe substantially solid or pasty in character, such as toothpowder, adental tablet or a dentifrice, that is a toothpaste (dental cream) orgel dentifrice. The vehicle of such solid or pasty oral preparationsgenerally contains dentally acceptable polishing material. Examples ofpolishing materials are water-insoluable sodium metaphosphate, potassiummetaphosphate, tricalcium phosphate, dihydrated calcium phosphate,anhydrous dicalcium phosphate, calcium pyrophosphate, magnesiumorthophosphate, trimagnesium phosphate, calcium carbonate, hydratedalumina, calcined alumina, aluminum silicate, zirconium silicate,silica, bentonite, and mixtures thereof. Other suitable polishingmaterial include the particulate thermosetting resins such as melamine-,phenolic, and urea-formaldehydes, and cross-linked polyepoxides andpolyesters. Preferred polishing materials include crystalline silicahaving particle sized of up to about 5 microns, a mean particle size ofup to about 1.1 microns, and a surface area of up to about 50,000cm²/gm., silica gel or colloidal silica, and complex amorphous alkalimetal aluminosilicate.

[0070] When visually clear gels are employed, a polishing agent ofcolloidal silica, such as those sold under the trademark SYLOID asSyloid 72 and Syloid 74 or under the trademark SANTOCEL as Santocel 100,alkali metal alumino-silicate complexes are particularly useful sincethey have refractive indices close to the refractive indices of gellingagent-liquid (including water and/or humectant) systems commonly used indentifrices.

[0071] Many of the so-called “water insoluble” polishing materials areanionic in character and also include small amounts of soluble material.Thus, insoluble sodium metaphosphate may be formed in any suitablemanner as illustrated by Thorpe's Dictionary of Applied Chemistry,Volume 9. 4th Edition. pp. 510-511. The forms of insoluble sodiummetaphosphate known as Madrell's salt and Kurrol's salt are furtherexamples of suitable materials. These metaphosphate salts exhibit only aminute solubility in water, and therefore are commonly referred to asinsoluble metaphosphates (IMP). There is present therein a minor amountof soluble phosphate material as impurities, usually a few percent suchas up to 4% by weight. The amount of soluble phosphate material, whichis believed to include a soluble sodium trimetaphosphate in the case ofinsoluble metaphosphate, may be reduced or eliminated by washing withwater if desired. The insoluble alkali metal metaphosphate is typicallyemployed in powder form of a particle size such that no more than 1% ofthe material is larger than 37 microns.

[0072] The polishing material is generally present in the solid or pastycompositions in weight concentrations of about 10% to about 99%.Preferably, it is present in amounts from about 10% to about 75% intoothpaste, and from about 70% to about 99% in toothpowder. Intoothpastes, when the polishing material is silicious in nature, it isgenerally present in amount of about 10-30% by weight. Other polishingmaterials are typically present in amount of about 30-75% by weight.

[0073] In a toothpaste, the liquid vehicle may comprise water andhumectant typically in an amount ranging from about 10%/o to about 80%by weight of the preparation. Glycerine, propylene glycol, sorbitol andpolypropylene glycol exemplify suitable huniectants/carriers. Alsoadvantageous are liquid mixtures of water, glycerine and sorbitol. Inclear gels where the refractive index is an important consideration,about 2.5-30% w/w of water, 0 to about 70% w/w of glycerine and about20-80% w/w of sorbitol are preferably employed.

[0074] Toothpaste, creams and gels typically contain a natural orsynthetic thickener or gelling agent in proportions of about 0.1 toabout 10, preferably about 0.5 to about 5% w/w. A suitable thickener issynthetic hectorite, a synthetic colloidal magnesium alkali metalsilicate complex clay available for example as Laponite (e.g. CP, SP2002, D) marketed by Laporte Industries Limited. Laponite D is,approximately by weight 58.00% SiO₂, 25.40% MgO, 3.05% Na₂O, 0.98% Li₂O,and some water and trace metals. Its true specific gravity is 2.53 andit has an apparent bulk density of 1.0 g/ml at 8% moisture.

[0075] Other suitable thickeners include Irish moss, iota carrageenan,gum tragacanth, starch, polyvinylpyrrolidone,hydroxyethylpropylcellulose, hydroxybutyl methyl cellulose,hydroxypropyl methyl cellulose, hydroxyethyl cellulose (e.g. availableas Natrosol), sodium carboxymethyl cellulose, and colloidal silica suchas finely ground Syloid (e.g. 244). Solubilizing agents may also beincluded such as humectant polyols such propylene glycol, dipropyleneglycol and hexylene glycol, cellosolves such as methyl cellosolve andethyl cellosolve, vegetable oils and waxes containing at least about 12carbons in a straight chain such as olive oil, castor oil and petrolatumand esters such as amyl acetate, ethyl acetate and benzyl benzoate.

[0076] It will be understood that, as is conventional, the oralpreparations are to be sold or otherwise distributed in suitablelabelled packages. Thus, a jar of mouthrinse will have a labeldescribing it, in substance, as a mouthrinse or mouthwash and havingdirections for its use: and a toothpaste, cream or gel will usually bein a collapsible tube, typically aluminium, lined lead or plastic, orother squeeze, pump or pressurized dispenser for metering out thecontents, having a label describing it, in substance, as a toothpaste,gel or dental cream.

[0077] Organic surface-active agents are used in the compositions of thepresent invention to achieve increased prophylactic action, assist inachieving thorough and complete dispersion of the active agentthroughout the oral cavity, and render the instant compositions morecosmetically acceptable. The organic surface-active material ispreferably anionic, nonionic or ampholytic in nature which does notdenature the antibody of the invention, and it is preferred to employ asthe surface-active agent a detersive material which imparts to thecomposition detersive and foaming properties while not denaturing theantibody. Suitable examples of anionic surfactants are water-solublesalts of higher fatty acid monoglyceride monosulfates, such as thesodium salt of the monosulfated monoglyceride of hydrogenated coconutoil fatty acids, higher alkyl sulfates such as sodium lauryl sulfate,alkyl aryl sulfonates such as sodium dodecyl benzene sulfonate, higheralkylsulfo-acetates, higher fatty acid esters of 1,2-dihydroxy propanesulfonate, and the substantially saturated higher aliphatic acyl amidesof lower aliphatic amino carboxylic acid compounds, such as those having12 to 16 carbons in the fatty acid, alkyl or acyl radicals, and thelike. Examples of the last mentioned amides are N-lauroyl sarcosine, andthe sodium, potassium, and ethanolamine salts of N-lauroyl, N-myristoyl,or N-palmitoyl sarcosine which should be substantially free from soap orsimilar higher fatty acid material. The use of these sarconite compoundsin the oral compositions of the present invention is particularlyadvantageous since these materials exhibit a prolonged marked effect inthe inhibition of acid formation in the oral cavity due to carbohydratesbreakdown in addition to exerting some reduction in the solubility oftooth enamel in acid solutions. Examples of water-soluble nonionicsurfactants suitable for use with antibodies are condensation productsof ethylene oxide with various reactive hydrogen-containing compoundsreactive therewith having long hydrophobic chains (e.g. aliphatic chainsof about 12 to 20 carbon atoms), which condensation products(“ethoxamers”) contain hydrophilic polyoxyethylene moieties, such ascondensation products of poly (ethylene oxide) with fatty acids, fattyalcohols, fatty amides, polyhydric alcohols (e.g. sorbitan monostearate)and polypropyleneoxide (e.g. Pluronic materials).

[0078] Surface active agent is typically present in amount of about0.1-5% by weight. It is noteworthy, that the surface active agent mayassist in the dissolving of the antibody of the invention and therebydiminish the amount of solubilizing humectant needed.

[0079] Various other materials may be incorporated in the oralpreparations of this invention such as whitening agents, preservatives,silicones, chlorophyll compounds and/or ammoniated material such asurea, diammonium phosphate, and mixtures thereof. These adjuvants, wherepresent, are incorporated in the preparations in amounts which do notsubstantially adversely affect the properties and characteristicsdesired.

[0080] Any suitable flavoring or sweetening material may also beemployed. Examples of suitable flavoring constituents are flavoringoils, e.g. oil of spearmint, peppermint, wintergreen, sassafras, clove,sage, eucalyptus, marjoram, cinnamon, lemon, and orange, and methylsalicylate. Suitable sweetening agents include sucrose, lactose,maltose, sorbitol, xylitol, sodium cyclamate, perillartine, AMP(aspartyl phenyl alanine, methyl ester), saccharine, and the like.Suitably, flavor and sweetening agents may each or together comprisefrom about 0.1% to 5% more of the preparation.

[0081] In the preferred practice of this invention an oral compositionaccording to this invention such as mouthwash or dentifrice containingthe composition of the present invention is preferably applied regularlyto the gums and teeth, such as every day or every second or third day orpreferably from 1 to 3 times daily, at a pH of about 4.5 to about 9,generally about 5.5 to about 8, preferably about 6 to 8, for at least 2weeks up to 8 weeks or more up to a lifetime.

[0082] The compositions of this invention can be incorporated inlozenges, or in chewing gum or other products, e.g. by stirring into awarm gum base or coating the outer surface of a gum base, illustrativeof which may be mentioned jelutong, rubber latex, vinylite resins, etc.,desirably with conventional plasticizers or softeners, sugar or othersweeteners or such as glucose, sorbitol and the like.

[0083] Another important form of the invention is a immunogeniccomposition based on the synthetic peptide antigens and suitableadjuvant delivered by nasal spray, orally or by injection to produce aspecific immune response against the antigen thereby reducingcolonisation of P. gingivalis and reducing virulence thereby preventingdisease. Unlike whole P. gingivalis cells or other previously preparedantigens, the peptide antigens described herin are safe and effectiveantigens for the preparation of a vaccine for the prevention of P.gingivalis associated periodontal disease. Additionally, according tothe present invention, antigenic peptide produced may be used togenerate P. gingivalis antisera useful for passive immunization againstperiodontal disease and infections caused by P. gingivalis.

[0084] The following examples are further illustrative of the nature ofthe present invention, but it is understood that the invention is notlimited thereto. All amounts and proportions referred to herein and inthe appended claims are by weight unless otherwise indicated.

EXAMPLE 1

[0085] The identification of the proteinase active site and adhesinbinding motifs was facilitated by the cloning and characterisation ofthe second gene encoding an Arg-specific proteinase of P. gingivalisW50.

[0086] Materials

[0087] O-Benzotriazole-N,N,N′,N′-tetramethyluronium hexafluorophosphate(HBTU), 1hydroxybenzotriazole (HOBt), diisopropylethylamine (DIPEA),N,N-dimethylformamide (DMF), piperidine, trifluoroacetic acid (TFA) and9-fluorenylmethoxycarbonyl (Fmoc)-protected amino acids were obtainedfrom Auspep Pty Ltd (Melbourne, Australia). Triisopropylsilane (TIPS)and ethanedithiol (EDT) were obtained from Aldrich (New South Wales,Australia). 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) was obtained fromSigma Chemical Company (New South Wales, Australia). Phenol and diethylether were obtained from BDH (Poole, UK). Unless otherwise statedchemicals were of peptide synthesis grade or its equivalent.

[0088] Bacterial Strain and Growth Conditions

[0089] Lyophilized cultures of Porphyromonas gingivalis W50 were kindlyprovided by Professor P. Marsh (PHLS, Centre for Applied Microbiologyand Research, Wiltshire, UK). P. gingivalis W50 was grown anaerobically(Bhogal et al., 1997) and Escherichia coli JM109 and LE392 strains weregrown following the procedures previously described (Slakeski et al.,1996).

[0090] Purification of the 50 kDa Arg-Specific Proteinase

[0091]P. gingivalis W50 was grown in batch culture (5 L) and harvestedat late logarithmic phase by centrifugation (5,000×g, 20 min, 4° C.).Cells were washed once with 150 ml TC buffer (20 mM Tris-HCl pH 7.4 and5 mM CaCl₂) containing 50 mM NaCl and sonicated as described previously(Bhogal et al., 1997). The sonicate was centrifuged (100,000×g, 30 min,4° C.) and the supernatant filtered (0.22 οm) prior to anion-exchangeFPLC. The sonicate was applied to an anion-exchange column (Hiload XK16/10 Q Sepharose, Pharmacia-LKB) cooled to 4° C., in multipleinjections using a 50 ml superloop (Pharmacia-LKB). The sonicate waseluted using a linear gradient from 0-100% buffer B over 90 min at aflow rate of 2.0 ml min⁻¹. Absorbance was monitored at 280 nm andelutant collected at 4° C. in 6 ml fractions using a Frac 100 fractioncollector (Pharmacia-LKB). Buffer A was TC buffer containing 50 mM NaCland buffer B was TC buffer containing 500 mM NaCl. Fractions wereanalysed for proteolytic and amidolytic activity using azocasein(A-2765, Sigma Chemical Co. St Louis, Mo.), benzoyl-L-Arg-p-nitroanilide(Bz-L-Arg-pNA, Sigma) and benzyloxycarbonyl-L-Lys-p-nitroanilide(z-L-Lys-pNA, Calbiochem, Melbourne, Australia) as described previously(Bhogal et al., 1997) except that fractions were pre-incubated with 10mM cysteine for 10 min at 25° C. before the addition of substrate. Forthe amidolytic assays absorbance was monitored at 410 nm as previouslydescribed (Bhogal et al., 1997) and the amidolytic activity expressed asU where U=μmol substrate converted min⁻¹ at 25° C. Anion-exchangefractions eluting between 160-246 mM NaCl containing the highest ratioof Arg-specific to Lys-specific activity were, washed and concentratedin TC buffer containing 150 mM NaCl using a centripep and centricon 10concentrators (Amicon) and applied to a gel filtration column (Superose12, HR 10/30, Pharmacia-LKB) using TC buffer containing 150 mM NaCl at aflow rate of 0.3 ml min⁻¹. Absorbance was monitored at 280 nm andfractions collected at 4° C. using a Frac 100 fraction collector. TheM_(r) values of eluant peaks were determined using gel filtrationmolecular mass standards (Pharmacia-LKB). The peak eluting at 50 kDacontaining only Arg-specific amidolytic activity was washed in TC buffercontaining 50 mM NaCl using a centricon-10 concentrator (Amicon) andapplied to a Mono Q (HR 5/5) anion-exchange column using a 5 ml loop andeluted using a linear gradient of O-100% buffer B at a flow rate 1.0 mlmin⁻¹. Buffer A was TC buffer containing 150 mM NaCl, buffer B was TCbuffer containing 500 mM NaCl. Absorbance was monitored at 280 nm andfractions collected at 4° C. using a Frac 100 fraction collector.

[0092] SDS-PAGE, Protein Transblot and N-terminal Sequence Analysis

[0093] SDS-PAGE was performed using a Mini protean II electrophoresissystem (Biorad) with 12% (w/v), 1 mm separating gels, overlaid with 5%stacking gels (Laemmli, 1970) and proteins transblotted and N-terminallysequenced using the procedures previously described (Bhogal et al.,1997).

[0094] Cloning and Nucleotide Sequence Analysis

[0095] The P. gingivalis W50 LambdaGEM®-12 genomic library, describedpreviously (Slakeski et al., 1996) was screened using syntheticoligonucleotides derived from the nucleotide sequence of prtR (Slakeskiet al., 1996) corresponding to the N-terminal sequence of PrtR45.Oligonucleotide probes were 5′ end-labelled using γ³²P ATP and T4polynucleotide kinase. Approximately 1.5×10⁴ phage were screened bylifting onto Nylon membrane filters and hybridising with radiolabelledoligonucleotides overnight in hybridisation buffer: 6×SSC (SSC is 15 mMsodium citrate, 150 mM NaCl pH 8.0), 0.25% SDS, 5× Denhardt's solution(Sambrook et al., 1989) and 100 μg/ml salmon sperm DNA at 49° C. Filterswere washed extensively in a solution of 2×SSC containing 0.1% SDS (w/v)at 49° C. Phage from positively-hybridising plaques were purified usingstandard procedures (Sambrook et al., 1989). Phage DNA was digested withEco72 I and the resulting fragments ligated into Sma I-BAP pUC18(Pharmacia, Sydney, Australia) which was used to transform E. coli JM109using the heat shock procedure (Sambrook et al., 1989). Double-strandedtemplate DNA was sequenced as described previously (Slakeski et al.,1996).

[0096] PCR was used to amplify a 991 bp fragment containing the internalEco 721 site encoded by prtRII using the two oligonucleotide primers5′-CGGCTTCCGTAAAGTC-3′ (forward primer identical to bases 657-672 ofPrtRII sequence) and 5′-TGGCTACGATGACGATCATACGAC-3′ (reverse primer with96% identity to bases 1624-1647 of PrtRII). The PCR was carried out in afinal volume of 100 μl and each reaction mixture contained 100 ng P.ginigivalis W50 genomic DNA, 0.2 mM dNTPs, 1.5 mM MgCl₂, 100 pmol ofeach primer. 20 mM Tris-HCl, pH 8.4, 50 mM KCl and 2.5 U Taq DNAPolymerase (Gibco BRL). The reaction mixture was heated at 95° C. for 3min and then subject to 25 cycles of DNA denaturation at 95° C. for 30s, primer annealing at 40° C. for 1 min and extension at 72° C. for 2min. Following cycling, the reaction mixture was finally heated at 72°C. for 5 min. Amplified DNA was purified using a PCR Spinclean Kit(Progen) and sequenced across the Eco 72I site in both directions.

[0097] Purification of High Molecular Mass Complexes of Arg-Specific andLys-Specific Proteinases and Adhesins (PrtR-PrtK Complexes)

[0098] The high molecular mass, cell-associated proteinase-adhesincomplexes (PrtR-PrtK complexes) of P. gingivalis W50 were purified usinga combination of anion-exchange, gel filtration and Arg-sepharoseaffinity chromatography from a cell sonicate as described previously(Bhogal et al., 1997). The complexes were characterised using SDS-PAGE,transblotting and sequence analysis and assayed for enzymic activityusing Bz-L-Arg-pNA and Z-L-Lys-pNA substrates (Bhogal et al., 1997).

[0099] Solid-Phase Peptide Synthesis

[0100] Peptides were synthesised manually using standard Fmocsolid-phase peptide synthesis protocols. The peptides were assembled asthe carboxyamide form using Fmoc-Pal-Peg-PS resin (PerSeptive BiosystemsInc. Framingham, Mass.). Coupling was accomplished with HBTU/HOBtactivation using 4 equiv of Fmoc-amino acid and 6 equiv of DIPEA. TheFmoc group was removed by 2% v/v DBU in DMF containing 2% v/vpiperidine. Cleavage of peptides from the resin support was performedusing TFA:phenol:TIPS:EDT:water (92:2:2:2:2) cleavage cocktail for 2.5hours. After cleavage the resin was removed by filtration and thefiltrate concentrated to approximately 1 ml under a stream of nitrogen.After the peptide products were precipitated in cold ether, they werecentrifuged and washed three times. The peptide precipitate was thendissolved in 10 ml of water containing 0.1% v/v TFA and insolubleresidue removed by centrifugation.

[0101] Purification of synthesized peptides was performed using aBrownlee C18 Aquapore ODS column (250×100 mm) installed in a Waters HPLCsystem. Chromatograms were developed at a flow rate of 5.0 ml min⁻¹using 0.1% v/v TFA in wvater (solvent A) and 0.1% v/v TFA in 90% aqueousacetonitrile (solvent B). Peptides were eluted with a gradient of 10-30%solvent B over 40 min. Analytical HPLC was carried out using a BrownleeC8 Aquapore RP-300 column (220×4.6 mm) installed in a Applied BiosytemsHPLC system. Chromatograms were developed using solvent A and solvent Bat a flow rate of 1.0 ml min⁻¹ and a 0-100% linear gradient of solvent Bover 30 min. Material eluted from the columns was monitored byabsorbance at 214 nm. Peptides were analysed by mass spectrometry usinga PerSeptive Biosystems Voyager DE MALDI-TOF.

[0102] Competitive Binding Assay

[0103] Wells of flat-bottomed polyvinyl microtitre plates (Microtitre,Dynatech Laboratories, Va.) were coated overnight at 4° C. using asolution (5 mg/ml) of the adhesin binding motif (ABM) peptide in 0.1Mphosphate buffered saline, pH 7.4, containing 0.1% v/v Tween 20 (PBST)and 0.1% w/v sodium azide. After removal of the coating solution, 2% w/vskim milk powder in PBST was added to block the remaining uncoatedplastic for 1 hour at room temperature and then washed (4×PBST). Asolution (1 mg/ml) of the PrtR-PrtK proteinase-adhesion complex(inactivated with 1 mM TLCK) was incubated with known concentrations ofABM peptide, control peptide and casein for 1 hour and then transferredto the microtitre plates coated with the ABM peptide. Followingincubation for 2 hours at 37° C. the plates were washed (5×PBST). A1/10,000 dilution of rabbit anti-PrtR-PrtK antisera in PBST containing1% w/v skim milk powder was then added to the washed wells and incubatedfor 2 hours at 37° C. Bound antibody was detected by incubation withhorseradish peroxidase-conjugated goat immunoglobulin (Ig) directedagainst rabbit Ig (BioRad, Richmond, Calif.) for 1.5 hours at 37° C.After washing (5×PBST), substrate (0.4 mM 3,3′,5,5′-tetramethylbenzidinein 0.1M sodium acetate/citric acid buffer containing 0.004% v/v hydrogenperoxide) was added and colour development was stopped by addition of 2MH₂SO₄. Optical density (O.D.) at 450 nm was measured using a BioRadmicroplate reader model 450.

[0104] Results

[0105] PrtRII50 Arg-Specific Proteinase Purification andCharacterisation

[0106] The P. gingivalis W50 cell sonicate contained 0.36 mg ml⁻¹protein and 2.4 and 1.1 μmol min⁻¹ mg protein⁻¹ activity with 1.0 mMBz-L-Arg-pNA and z-L-Lys-pNA as substrates respectively at 25° C. Thesonicate was subjected to Q-sepharose anion exchange FPLC andproteolytic/amidolytic activity eluting between 160-246 mM NaCl wascollected and concentrated using a centripep and centricon-10concentrator (Amicon, Sydney, Australia). This fraction represented theleading edge of the main peak of proteolytic/amidolytic activity andcontained the highest ratio of Arg-specific activity to Lys-specificactivity. After concentration, the fraction was applied to a Superose 12gel filtration column (FIG. 6). Arg- and Lys-specific activity wasassociated with the high molecular mass eluting material correspondingto peaks with M_(r) values of 0.6-2.0×10⁶ Da and 300 kDa as reportedpreviously (Bhogal et al., 1997). However, a lower molecular mass peakof 50 kDa was also observed, which displayed only Arg-specific activityand this peak was collected for further purification. The 50 kDa gelfiltration peak was applied to a Mono Q anion exchange column and uponapplication of a NaCl gradient the Arg-specific activity eluted in adistinct peak at a NaCl concentration of 200 mM NaCl with a 28-foldpurification over the original crude sonicate. The peak containingArg-specific activity was subjected to SDS-PAGE which confirmed a single50 kDa protein band (FIG. 7). The 50 kDa band was transblotted andsubjected to N-terminal sequence analysis which provided the amino acylsequence

[0107] YTPVEEKENGRMIVIVPKKYEEDIED. The specificity of the 50 kDaproteinase for arginyl residues was confirmed by the enzyme cleavingBz-L-Arg-pNA but not z-L-Lys-pNA. The Arg-specific 50 kDa enzyme wasactivated by thiols (particularly cysteine), not inhibited by the serineproteinase inhibitors, phenylmethyl sulfonyl fluoride or4-(2-aminoethyl)-benzenesulfonyl fluoride but inhibited bysulphydryl-directed reagents, leupeptin and EDTA at similarconcentrations to that which inhibited the PrtR45 (Bhogal et al., 1997).Inhibition with EDTA could be reversed by the addition of excess Ca²⁺and the pH optimum of the enzyme was 8.0 with minimal activity below pH6.0.

[0108] Molecular Cloning and Sequence Analysis of the prtRII Gene

[0109] Screening of the P. gingivilis genomic library usingoligonucleotide probes specific for the N-terminus of PrtR45 identifiedseveral positive clones. The DNA from these clones was extracted andsubjected to Southern analysis to identify those containing the 12 kbBamH I fragment previously proposed to correspond to the gene encodingthe second Arg-specific proteinase (Slakeski et al., 1996). Lambda clone18, containing a 12 kb BamH I fragment was chosen for further analysisand DNA was isolated from this clone and digested with Eco72 I andrandomly cloned into plasmid Sma I-BAP pUC 18. Adjacent 3.3 and 1.2 kbEco72 I genomic fragments were sequenced in both directions to generatethe entire prtRII nucleotide sequence (Genebank Accession No. AF007124).A 991 bp PCR fragment was generated and sequenced to confirm thesequence encompassing the internal Eco72 I site.

[0110] The prtRII ORF comprises 2208 bp (736 a.a. residues) and encodesa preproprotein consisting of a putative leader sequence and aprofragment followed by the mature Arg-specific proteinase (507 a.a.residues) containing the exact N-terminal amino acyl sequence obtainedfor the purified 50 kDa enzyme (PrtRII50). The N-terminal sequence ofthe mature protein, like PrtR45, is immediately preceded by an Argresidue in the profragment.

[0111] The prtRII gene exhibits a high degree of similarity with the 5′two fifths of the prtR gene which encodes PrtR45 and its associatedadhesins (FIGS. 8 & 9). A comparison of the two translated sequencesshows an overall similarity of 76% and 80% for the translatedpreprofragment and the proteinase domain, respectively. The prtRIIhowever, does not encode any of the C-terminal haemagglutinin/adhesindomains encoded by the prtR and prtK genes being consistent with thefinding that the purified PrtRII50 proteinase was not associated withadhesins. The M_(r) of the PrtRII50 mature proteinase deduced from thetranslated prtRII gene sequence is 55.6 kDa which is consistent with the50 kDa obtained by SDS-PAGE (FIG. 7) and is slightly larger than thededuced M_(r) of 53.9 kDa for PrtR45 (Bhogal et al., 1997).

[0112] The sequence alignment of the deduced amino acyl sequence ofPrtRII50 with the PrtR45 Arg-specific proteinase and the PrtK48Lys-specific proteinase (Slakeski et al., 1996; Bhogal et al., 1997)shows that PrtRII50 displays high sequence similarity (97.5% identity)to the adhesin-associated PrtR45 proteinase except for the C-terminal 80amino acyl residues (FIG. 8). In fact, this C-terminal 80 residuesequence of PrtRII50 is similar (47% identity) to the C-terminal 80residues of the PrtR27 adhesin domain, the last domain of the PrtR (FIG.9). In contrast to the high sequence identity of the PrtRII50 and PrtR45proteinases, there is lower overall similarity (25% identity) betweenthe two adhesin-associated PrtR45 and PrtK48 proteinases except aroundthe C-terminal region where themotif—GEPNPYQPVSNLTATTQGQKVTLKWDAPSTK—(underlined in FIG. 8) is almostidentical in both proteinases but is absent in PrtRII50. Similar motifsalso occur in the PrtR44, PrtR17, PrtK39 and PrtK44 adhesin domains ofPrtR and PrtK (Table 1 ABM1 peptides), which have led us to propose thatthis motif is an adhesin-binding motif involved in the association ofthe PrtR and PrtK proteinases and adhesins into large complexes.

[0113] Binding of the PrtR-PrtK Complex to a Synthethic PeptideCorresponding to a Putative Adhesin Binding Motif

[0114] A peptide (ABM1 [R45]) corresponding to the proposed adhesinbinding motif PYQPVSNLTATTQGQKVTLKWDAPSTK, was synthesised and used tomeasure binding of the PrtR-PrtK complex. Specific binding ofTLCK-inactivated PrtR-PrtK complex to the ABM peptide was demonstratedin a competitive binding assay where a 5-100 fold molar excess of theABM peptide in solution was required to inhibit binding of the complexto the ABM peptide adsorbed onto the microtitre plate (FIG. 10). Acontrol peptide, FNGGISLANYTGHGSETAWGT corresponding to residues 428-448of PrtR45, as well as casein did not inhibit the binding of theTLCK-inactivated PrtR-PrtK complex to the adsorbed ABM peptide. Theanti-PrtR-PrtK antisera did not bind to the ABM peptide in the absenceof the PrtR-PrtK complex. The inactivation with TLCK ensured that thecomplex was not binding to the peptide through the active sites of theproteinases. This was also confirmed by lack of binding of the PrtR-PrtKcomplex to casein and a non-specific peptide of similar size and lysinecontent to the ABM peptide but of unrelated sequence. These resultsdemonstrating specific binding of the TLCK-inactivated PrtR-PrtK complexto the ABM peptide therefore are consistent with the proposed role ofthis conserved motif in the association of the PrtR and PrtK proteinasesand adhesins into large complexes.

[0115] Discussion

[0116] Using a P. gingivalis W50 cell sonicate we have purified andcharacterised a second cell-associated, Arg-specific, calcium-stabilizedcysteine proteinase that is almost identical to the previouslycharacterised Arg-specific cysteine proteinase PrtR45 (Bhogal et al.,1997). However, despite the almost identical enzymic characteristics andinhibitor/activator profile to PrtR45 the second enzyme exhibits anumber of key differences. Firstly, the second enzyme designatedPrtRII50. is a discrete enzyme not associated with adhesins. TheArg-specific cysteine proteinase, PrtR45, is a 45 kDa component of alarge multi-protein complex of Arg- and Lys-specific proteinases andadhesins (Bhogal et al., 1997). Secondly, PrtRII50 is slightly largerthan PrtR45 on SDS-PAGE (M_(r)50 kDa) and thirdly there are four aminoacid substitutions in the first 25 N-terminal residues of PrtRII50.PrtRII50 has a Glu at position 8 instead of Gln, a Pro at position 17instead of Ala, a Glu at position 22 instead of Gly and a Glu atposition 25 instead of the Lys in PrtR45 (FIG. 8). These differences insize and the N-terminal amino acyl sequence were confirmed with thecloning and sequence analysis of the gene prtRII encoding the secondArg-specific proteinase.

[0117] The deduced amino acid sequence of the prtRII gene exhibits 98%identity with that of the recently reported rgpB gene from P. gingivalisATCC 33277 (Nakayama, 1997) suggesting that both genes represent thesame locus in two different strains. However, the sequence for themature proteinase of the rgpB gene does not contain three of theN-terminal amino acyl substitutions found in the prtRII gene product andonly has the Gln→Glu substitution at position 8. The substitutions atpositions 17, 22 and 25 found in PrtRII50. that enabled the gene productto be unequivocally differentiated by N-terminal sequence analysis fromthe mature PrtR45 proteinase of the prtR (rgpA), were not found in thergpB. In the current study the differences in N-terminal sequence andsize of the mature proteinases enabled the differentiation of thediscrete 50 kDa Arg-specific proteinase (PrtRII50) from the 45 kDaArg-specific proteinase (PrtR45) found associated with adhesins. Theassignment of the two proteinases (PrtR45 and PrtRII50) to the two genes(prtR and prtRII respectively) has enabled identification of a conservedmotif in the two adhesin-associated proteinases (PrtR45 and PrtK48) notfound in the discrete PrtRII50. As the conserved motif was also found inseveral adhesins of the prtR and prtK we propose that it is an adhesinbinding motif involved in association of the prtR and prtK proteinasesand adhesins into large complexes. This proposition is supported by thedemonstration that a synthetic peptide corresponding to the conservedmotif specifically binds to the TLCK-inactivated PrtR-PrtK complex.

[0118] The identification of the conserved motif PVXNLT . . . LKWXAP inthe adhesin binding motif 1 led us to propose that the complementarymotif would be hydrophobic and negatively charged. Repeat motifs thatwere therefore hydrophobic and contatined negative residues wereselected for synthesis. eg. TATTFEEDGVA (ABM 2, Table 1) and WKTIDADGDG(ABM 5. Table 1). Other motifs selected for study were other repeatedmotifs of hydrophobic and/or charged and/or neutral polar residues eg.VYRDGTKIKE (ABM 2, Table 1), WEIRTVDLPAGTKYV (ABM 4, Table 1) andEFAPVQNLTGSA (ABM 6, Table 1).

[0119] On further examination of the alignment of the deduced amino acylsequence of PrtRII50 with the catalytic domains of the PrtR45Arg-specific proteinase and the PrtK48 Lys-specific proteinase somefurther interesting areas of similarity were revealed (FIG. 8). Althoughthese three cysteine proteinases from P. gingivalis have no similaritywith any of the other known families of cysteine proteinases it ispossible to speculate on the identity of the catalytic residues sinceonly one His residue and two Cys residues are conserved in the threesequence-related enzymes. The catalytic Cys, His dyad of these enzymestherefore is likely to consist of H¹⁴⁰ of PrtRII50, the only conservedHis in the three proteinases. The catalytic Cys is also likely to be oneof the two conserved cysteinyl residues C⁴⁷³ and C⁴⁸⁴ in the threesequence-related proteinases.

EXAMPLE 2

[0120] Synthesis of Proteinase Active Site and Adhesin Binding MotifPeptides and Testing in a Murine Lesion Model.

[0121] The following peptides representative of the protease activesites and each adhesin binding motif listed in Table 1 were synthesised,conjugated and tested in the murine lesion model (Table 2). TABLE 2Origin and amino acid sequence of synthesised peptides Amino acidsequence Origin (single letter code) Abbreviation Proteinase Active SitePeptides PrtR45 (426-446) FNGGISLANYTGHGSETAWGT PAS1 (R45) PrtK48(432-453) LNTGVSFANYTAHGSETAWADP PAS1 (K48) Adhesion Binding MotifPeptides PrtR45 (664-689) PYQPVSNLTATTQGQKVTLKWDAPSTK ABM1 (R45) PrtK39(1580-1608) SYTYTVYRDGTKIKEGLTATTFEEDGVAA ABM2 (K39) PrtR44 (939-971)VTLKWDAPNGTPNPNPNPNPNPNPGTTTLSESF ABM3 (R44) PrtK44 (1296-1315)WIERTVDLPAGTKYVAFRHY ABM4 (K44) PrtR15 (1154-1169) PAEWTTIDADGDGQGW ABM5(R15) PrtR44 (919-938) EGSNEFAPVQNLTGSAVGQK ABM6 (R44) Control PeptidePrtR27 (1432-1463) ANEAKVVLAADNVWGDNTGYQFLLDADHNTFG Control peptide

[0122] Materials

[0123] Unless otherwise stated chemicals were of peptide synthesis gradeor its equivalent. O-Benzotriazole-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (HBTU), 1hydroxybenzotriazole (HOBt),diisopropylethylamine (DIPEA), N,N-dimethylformamide (DMF), piperidine,trifluoroacetic acid (TFA) and 9-fluorenylmethoxycarbonyl (Fmoc)protected amino acids were obtained from Auspep Pty Ltd (Melbourne,Australia). Triisopropylsilane (TIPS) and ethanedithiol (EDT) wereobtained from Aldrich (New South Wales, Australia).1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) was obtained from SigmaChemical Company (New South Wales, Australia). Phenol and diethyl etherwere obtained from BDH (Poole, UK).

[0124] Solid-Phase Peptide Synthesis

[0125] Peptides were synthesised manually or using a 431A ABI peptidesynthesiser. Standard solid-phase peptide synthesis protocols for Fmocchemistry were used throughout. Peptides were assembled as thecarboxyamide form using Fmoc-Pal-Peg-PS resin (PerSeptive BiosystemsInc., Framingham, Mass.). Coupling was accomplished with HBTU/HOBtactivation using 4 equiv of Fmoc-amino acid and 6 equiv of DIPEA. TheFmoc group was removed by 2% v/v DBU in DMF containing 2% v/vpiperidine. Cleavage of peptides from the resin support was performedusing TFA:phenol:TIPS:EDT:water (92:2:2:2:2) cleavage cocktail for 2.5hours or 4 hours depending on the arginine content of the peptide. Aftercleavage the resin was removed by filtration and the filtrateconcentrated to approximately 1 mL under a stream of nitrogen. After thepeptide products were precipitated in cold ether, they were centrifugedand washed three times. The peptide precipitate was then dissolved in 5to 10 mL of water containing 0.1% v/v TFA and insoluble residue removedby centrifugation.

[0126] Synthesis of S-Acetylmercaptoacetic acid Peptides

[0127] Resins bearing peptides were swollen in DMF and the N-terminalFmoc group removed by 2% v/v DBU in DMF containing 2% v/v piperidine.S-Acetylmercaptoacetic acid (SAMA) group was introduced onto theN-terminal amino group using 5 equiv of SAMA-OPfp and 5 equiv of HOBt.The reaction was monitored by the trinitrobenzene sulphonic acid (TNBSA)test. When a negative TNBSA test was returned the resin was washed(5×DMF, 3×DCM and 3×diethyl ether). The resin was dried under vacuum andthe SAMA-peptides cleaved from the resin support as described above.

[0128] Peptide Purification

[0129] Purification of synthesized peptides was performed using aBrownlee C18 Aquapore ODS column (250×100 mm) installed in a Waters HPLCsystem. Chromatograms were developed at a flow rate of 5 mL/min using0.1% v/v TFA in water (solvent A) and 0.1% v/v TFA in 90% aqueousacetonitrile (solvent B) as the limit buffer. Peptides were eluted witha gradient of 10-30% solvent B formed over 40 min. Analytical HPLC wascarried out using a Brownlee C8 Aquapore RP-300 column (220×4.6 mm)installed in a Applied Biosytems HPLC system. Chromatograms weredeveloped using solvent A and solvent B at a flow rate of 1 mL/min and a0-100% linear gradient of solvent B formed over 30 min. Material elutedfrom the columns was detected by determining the absorbance at 214 nm.Peptide fractions were pooled and lyophilised. Peptides were analysed bymass spectrometry using a PerSeptive Biosystems Voyager DE MALDI-TOF.

[0130] Conjugation of SAMA-Peptides to Diphtheria Toxoid

[0131] Diphtheria toxoid (DT) was obtained from Dr I. Barr (CSL Pty.Ltd. Melbourne, Australia) which contained 9 equivalent amino groups per62 kDa molecule. To a solution containing 10 mg/mL of DT inphosphate-buffered saline (0.1M sodium phosphate, 0.9% NaCl; pH 7.4) wasadded 0.1 mL of a 1% w/v solution m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) in DMF. After 30 mins unreactedMBS was removed and NIBS modified DT collected by gel filtration using aPD10 column (Pharmacia, NSW, Australia) equilibrated in conjugationbuffer (0.1M sodium phosphate, 5 mM EDTA; pH 6.0). Purified SAMA-peptide(1.3 μmole) was dissolved in 200 μL 6M guanidine HCl containing 0.5MTris; 2 mM EDTA, pH6 and diluted with 800 μL MilliQ water anddeprotected in-situ by addition of 25 μL of 2M NH₂OH (40 equiv)dissolved in MilliQ water. The collected MBS-DT was immediately reactedwith deprotected SAMA-peptide and stirred for one hour at roomtemperature. The peptide-DT conjugate was separated from unreactedpeptide by gel filtration using a PD10 column equilibrated in PBS pH 7.4and lyophilised. The reaction was monitored using the Ellmans test. Theconjugation yields of SAMA-peptides to MBS-DT ranged from 34% to 45%indicating that 3 to 4 peptides were coupled per DT molecule.

[0132] Immunization and Murine Lesion Model Protocols

[0133] BALB/c mice 6-8 weeks old were immunised subcutaneously witheither 50 μg of the peptide-DT conjugate. 50 μg of DT or 2×10⁹ formalinkilled cells of Porphyromonas gingivalis strain 33277 emulsified incomplete Freund's adjuvant (CFA). After 30 days the mice were injectedsubcutaneously with antigen (either 50 μg of the peptide-DT conjugate,50 μg of DT or 2×10⁹ formalin killed cells of Porphyromonas gingivalisstrain 33277) emulsified in incomplete Freund's adjuvant (IFA) and thenbled from the retrobulbar plexus 12 days later. All mice were challengedwith 8×10⁹ cells of P. gingivalis (200 μL) by subcutaneous injection inthe abdomen and weighed and lesion size measured over 10 days. Lesionsizes are expressed as mm² and were statistically analysed using aKruskal-Wallis one-way ANOVA and Mann-Whitney U-Wilcoxon rank sum Wtest.

[0134] The peptide-DT conjugates were used to immunise BALB/c mice toevaluate their efficacy in protecting against Porphyromonas gingivalischallenge in the murine lesion model. FIG. 6 shows that mice that wereimmunised with the carrier protein diphtheria toxoid alone had similaraverage lesion sizes to the mice immunised with adjuvant alone(controls). This indicates that DT alone does not provide protectionagainst P. gingivalis and moreover that any protection provided bypeptide-DT conjugates was attributable to the immune response induced bythe peptide. The control peptide-DT conjugate did not provide protectionagainst P. gingivalis as the average lesion size was not significantlydifferent to that of mice immunised with DT or adjuvant alone(controls). Immunisation with both the proteinase active site peptidesconjugated to DT (PAS1(R45) and PAS1(K48)) Significantly reduced lesionsize resulting from P. gingivalis challenge relative to the DTcontrols(Table 3). All the adhesin binding motif peptides when used asimmunogens reduced lesion size however, only ABM1(R45), ABM2(K39) andABM3(R44) attained significance (p<0.05) with the number of animals used(Table 3).

[0135] The results demonstrate the effacacy of the PrtR-PrtK proteinaseactive site peptides and adhesin binding motif peptides when used asimmunogens in preventing challenge with P. gingivalis in the murinelesion model. These results therefore suggest that these peptides mayhave utility as vaccines in the prevention of P. gingivalis -associateddisease (e.g Periodontitis) in humans.

[0136] Antisera against the PAS1 peptides inhibited both Arg- andLys-specific proteolytic activity which therefore may explain theexcellent protection conferred by immunisation with these peptides. Theinhibition of proteolytic activity by the anti-PAS1 antisera suggeststhat these antibodies may have utility in a mouthwash, toothpaste orother intra-oral delivery vehicle to neutralise the P. gingivalisproteases and their damaging effects. Similarly, antisera against theadhesin binding motifs, particularly ABM1, ABM2 and ABM3 may haveutility in oral care products and pharmaceuticals to block adherence andtherefore colonisation of P. gingivalis. TABLE 3 Maximum Lesion size andsignificance of peptide-diphtheria conjugates. ABM1 AMB2 ABM3 ABM4 ABM5ABM6 PAS1 PAS1 Control FK DT^(d) (R45)-DT (K39)-DT (R44)-DT (K44)-DT(R15)-DT (R44)-DT (R45)-DT (K48)-DT peptide-DT 33277^(e) Maximum lesion33.59 10.42 12.63 12.27 18.83 14.79 15.22 10.46 9.28 36.61 13.78 size(mm²) ±18.77^(a) ±11.7 ±10.89 ±4.68 ±18.87 ±10.04 ±11.55 ±4.08 ±10.36±34.92 ±12.55 Significance^(b) — p < 0.05 p < 0.05 P < 0.05 N/S^(c)N/S^(c) N/S^(c) P < 0.05 p < 0.05 N/S^(c) p < 0.05

EXAMPLE 3

[0137] (1) Synthesis of Peptide Antigens and Multiple Constructs

[0138] The peptides of Table 1 were synthesized using standard Fmoc ortBoc synthesis strategies and multipeptide constructs were synthesizedusing the strategies outlined in FIGS. 1-5.

[0139] (2) Preparation of Antibodies

[0140] Serum antibodies were obtained by immunising horses, rabbits,sheep or dairy cows.

[0141] Immunizations were carried out using standard procedures. Theinitial immunisation was with a mixture of the antigen and Freund'sincomplete adjuvant. The antibodies could be recovered from the animalsserum or milk using standard procedures.

EXAMPLE 4

[0142] Methods for Using Antigenic Peptides in Diagnostic Immunoassays.

[0143] The P. gingivalis peptide antigens described herein can besynthesized for use as immunogens in vaccine formulations; and asantigens for diagnostic assays or for generating P. gingivalis -specificantisera of therapeutic and/or diagnostic value.

[0144] The peptides disclosed in Table 1 can be synthesized individuallyor chemically-linked using any one of a number of strategies well knownin the art. Examples of some strategies which can be used are set out inFIGS. 1-5. The peptides can be synthesized using one of the severalmethods of peptide synthesis known in the art including standard solidphase peptide synthesis using tertbutyloxycarbonyl amino acids (Mitchellet al., 1978, J. Org. Chem. 43:2845-2852), using9-fluorenylmethyloxycarbonyl amino acids on a polyamide support (Drylandet al., 1986, J. Chem. So. Perkin Trans. I, 125-137); by pepscansynthesis (Geysen et al., 1987, J. Immunol. Methods 03:259; 1984, Proc.Natl. Acad. Sci. USA 81:3998); or by standard liquid phase peptidesynthesis. Modification of the peptides or oligopeptides, such as bydeletion and substitution of amino acids (and including extensions andadditions to amino acids) and in other ways, maybe made so as to notsubstantially detract from the inmunological properties of the peptideor oligopeptide. In particular, the amino acid sequences of the antigensdescribed herein, may be altered by replacing one or more amino acidswith functionally equivalent amino acids resulting in an alterationwhich is silent in terms of an observed difference in thephysicochemical behaviour of the peptide, or oligopeptide or chimera.Functionally equivalent amino acids are known in the art as amino acidswhich are related and/or have similar polarity or charge. Thus, an aminoacid sequence which is substantially that of the amino acid sequencesdepicted in the Sequence Listing herein, refers to an amino acidsequence that contains substitutions with functionally equivalent aminoacids without changing the primary biological function of the peptide,oligopeptide or chimera.

[0145] Purified synthetic peptides may be used as antigens inimmunoassays for the detection of P. gingivalis -specific antiserapresent in the body fluid of an individual suspected of having aninfection caused by P. gingivalis. The detection of antigens or relatedpeptides in immunoassays, includes any immunoassay known in the artincluding, but not limited to, radioimmunoassay, enzyme-linkedimmunosorbent assay (ELISA), “sandwich” assay, precipitin reaction,agglutination assay, fluorescent immunoassay, andchemiluminescence-based immunoassay.

EXAMPLE 5

[0146] Methods and Compounds for Vaccine Formulations Related toSynthetic Peptide Antigens and Multipeptide Constructs.

[0147] This embodiment of the present invention is to provide peptideantigens of Table 1 to be used as immunogens in a prophylactic and/ortherapeutic vaccine for active immunization to protect against or treatinfections caused by P. gingivalis. For vaccine purposes, an antigen ofP. gingivalis comprising a synthetic peptide construct should beimmunogenic, and induce functional antibodies directed to one or moresurface-exposed epitopes on intact bacteria, wherein the epitope(s) areconserved amongst strains of P. gingivalis.

[0148] In one illustration of the invention, the dipeptide PAS1-PAS2construct (FIG. 4) having the properties desirable of a vaccine antigen,the dipeptide construct can be synthesized using the method describedherein in Example 3.

[0149] The synthetic peptide is included as the relevant immunogenicmaterial in the vaccine formulation, and in therapeutically effectiveamounts, to induce an immune response. Many methods are known for theintroduction of a vaccine formulation into the human or animal to bevaccinated. These include, but are not limited to, intradermal,intramuscular, intraperitoneal, intravenous, subcutaneous, ocular,intranasal, and oral administration. The vaccine may further comprise aphysiological carrier such as a solution, a polymer or liposomes; and anadjuvant, or a combination thereof.

[0150] Various adjuvants are used in conjunction with vaccineformulations. The adjuvants aid by modulating the immune response and inattaining a more durable and higher level of immunity using smalleramounts of vaccine antigen or fewer doses than if the vaccine antigenwere administered alone. Examples of adjuvants include incompleteFreund's adjuvant (ISA). Adjuvant 65 (containing peanut oil, mannidemonooleate and aluminum monostrearate), oil emulsions, Ribi adjuvant,the pluronic polyols, polyamines, Avridine, Quil A, saponin. MPL, QS-21,and mineral gels such as aluminum hydroxide, aluminum phosphate, etc.

[0151] Another embodiment of this mode of the invention involves theproduction of antigen-specific amino acid sequences as a hapten, i.e. amolecule which cannot by itself elicit an immune response. In such case,the hapten may be covalently bound to a carrier or other immunogenicmolecule which will confer immunogenicity to the coupled hapten whenexposed to the immune system. Thus, such a antigen-specific haptenlinked to a carrier molecule may be the immunogen in a vaccineformulation.

[0152] As an alternative to active immunization, immunization may bepassive. i.e. immunization comprising administration of purifiedimmunoglobulin containing antibody against synthetic peptides.

EXAMPLE 6

[0153] The following is an example of a proposed toothpaste formulationcontaining anti-peptide antibodies. Ingredient % w/w Dicalcium phosphatedihydrate 50.0 Glycerol 20.0 Sodium carboxymethyl cellulose 1.0 Sodiumlauryl sulphate 1.5 Sodium lauroyl sarconisate 0.5 Flavour 1.0 Sodiumsaccharin 0.1 Chlorhexidine gluconate 0.01 Dextranase 0.01 Goat serumcontaining anti-peptide Abs 0.2 Water balance

EXAMPLE 7

[0154] The following is an example of a proposed toothpaste formulation.Ingredient % w/w Dicalcium phosphate dihydrate 50.0 Sorbitol 10.0Glycerol 10.0 Sodium carboxymethyl cellulose 1.0 Sodium lauryl sulphate1.5 Flavour 1.0 Sodium saccharin 0.1 Sodium monofluorophosphate 0.3Chlorhexidine gluconate 0.01 Dextranase 0.01 Bovine serum containinganti-peptide Abs 0.2 Water balance

EXAMPLE 8

[0155] The following is an example of a proposed toothpaste formulation.Ingredient % w/w Dicalcium phosphate dihydrate 50.0 Sorbitol 10.0Glycerol 10.0 Sodium carboxymethyl cellulose 1.0 Lauroyl diethanolamide1.0 Sucrose monolaurate 2.0 Flavour 1.0 Sodium saccharin 0.1 Sodiummonofluorophosphate 0.3 Chlorhexidine gluconate 0.01 Dextranase 0.01Bovine milk 1 g containing anti-peptide Abs 0.1 Water balance

EXAMPLE 9

[0156] The following is an example of a proposed toothpaste formulation.Ingredient % w/w Sorbitol 22.0 Irish moss 1.0 Sodium Hydroxide (50%) 1.0Gantrez 19.0 Water (deionised) 2.69 Sodium Monofluorophosphate 0.76Sodium saccharine 0.3 Pyrophosphate 2.0 Hydrated alumina 48.0 Flavouroil 0.95 anti-peptide mouse monoclonal 0.3 sodium lauryl sulphate 2.00

EXAMPLE 10

[0157] The following is an example of a proposed liquid toothpasteformulation. Ingredient % w/w Sodium polyacrylate 50.0 Sorbitol 10.0Glycerol 20.0 Flavour 1.0 Sodium saccharin 0.1 Sodiummonofluorophosphate 0.3 Chlorhexidine gluconate 0.01 Ethanol 3.0 EquineIg containing anti-peptide Ab 0.2 Linolic acid 0.05 Water balance

EXAMPLE 11

[0158] The following is an example of a proposed mouthwash formulation.Ingredient % w/w Ethanol 20.0 Flavour 1.0 Sodium saccharin 0.1 Sodiummonofluorophosphate 0.3 Chlorhexidine gluconate 0.01 Lauroyldiethanolamide 0.3 Rabbit Ig containing anti-peptide-Ab 0.2 Waterbalance

EXAMPLE 12

[0159] The following is an example of a proposed mouthwash formulation.Ingredient % w/w Gantrez S-97 2.5 Glycerine 10.0 Flavour oil 0.4 Sodiummonofluorophosphate 0.05 Chlorhexidine gluconate 0.01 Lauroyldiethanolamide 0.2 Mouse anti-peptide monoclonal 0.3 Water balance

EXAMPLE 13

[0160] The following is an example of a proposed lozenge formulation.Ingredient % w/w Sugar   75-80 Corn syrup   1-20 Flavour oil   1-2 NaF0.01-0.05 Mouse anti-peptide monoclonal 0.3 Mg stearate   1-5 Waterbalance

EXAMPLE 14

[0161] The following is an example of a proposed gingival massage creamIngredient % w/w White petrolatum 8.0 Propylene glycol 4.0 Stear.ylalcohol 8.0 Polyethylene Glycol 4000 25.0 Polyethylene Glycol 400 37.0Sucrose monostearate 0.5 Chlorohexidine gluconate 0.1 Mouse anti-peptidemonoclonal 0.3 Water balance

EXAMPLE 15

[0162] The following is an example of a proposed chewing gumformulation. Ingredient % w/w Gum base 30.0 Calcium carbonate 2.0Crystalline sorbitol 53.0 Glycerine 0.5 Flavour oil 0.1 Mouseanti-peptide monoclonals 0.3 Water balance

[0163] It will be appreciated by persons skilled in the art thatnumerous variations and/or modifications may be made to the invention asshown in the specific embodiments without departing from the spirit orscope of the invention as broadly described. The present embodimentsare, therefore, to be considered in all respects as illustrative and notrestrictive.

[0164] References

[0165] Alexander, J., Sidney, J., Southwood, S., et al (1994).“Development of high potentcy universal DR-restricted helper epitopes bymodification of high affinity DR-blocking peptides.” Immunity 1:751-761.

[0166] Bhogal, P. S., Slakeski, N. & Reynolds, E. C. (1997).Characterization of a cell-associated, protein complex of Porphyromonasgingivalis W50 containing Arg- and Lys-specific cysteine proteinases andadhesins. Microbiology 143, 2485-2495.

[0167] Canne, L. E., Ferre-D'Amare, A. R., Burley, S. K., and Kent, S.B. H. (1995). “Total chemical synthesis of a unique transcriptionfactor-related protein: cMyc-Max.” J. A. Chem. Soc. 117: 2998-3001.

[0168] Druland, et. al. (1986). J. Chem. Soc. Perkin Trans. 1: 125-137.

[0169] Duncan, R., and Kopececk, J. (1980). “Degradation of side chainsof N-(2-hydroxypropyl)methacrylamide copolymers by lysosomal enzymes.”Biochem. Biophys. Res. Commun. 94: 284-290.

[0170] Geysen, H. M., Meleon, R. H., and Barteling, S. J. (1984). “Useof peptide synthesis to probe viral antigens for epitopes to aresolution of a single amino acid.” Proc. Natl. Acad. Sci. USA. 81:3998.

[0171] Geysen, H. M., Rodda, S. J., Mason, T. J., et al. (1987).“Strategies for epitope mapping using peptide synthesis.” J. Immunol.Methods. 102: 259.

[0172] Hammer, J., Valsasnini, P., Tolba, K., Bolin, D., Higelin, J.,Takacs, B., and Sinigaglia, F. (1993). “Promiscuous and allele-specificanchors in HLA-DR-binding peptides.” Cell 74: 197-203.

[0173] Kaumaya. P. T. P., Kobs-Conrad. S., and DiGeorge, A. M. (1994).Synthetic peptide vaccines: Misconceptions and problems, strategies andprospects Innovation and Perspectives in Solid Phase Synthesis. R.Epton. Kingswinford, Mayflower: 279-292.

[0174] Liu, C. F. a. T., J. P. (1994). “Peptide ligation strategywithout use of protectecing groups.” Proc. Natl. Acad. Sci. USA 91:6584-6588.

[0175] Lu, Y. A., Clavijo, P., Galantino, M., Shen, Z. Y., and Tam, J.P. (1991). “Chemically unambiguous peptide immunogen: Preparation,orientation and antigenicity of purified peptide cinjugated to themultiple antigen peptide system.” Mol. Immunol. 28(6): 623-630.

[0176] Mitchell., e. a. (1978). J. Org. Chem. 43: 2845-2852.

[0177] Nakayama, K. (1997). Domain-specific rearrangement between thetwo Arg-gingipain-encoding genes in Porphyromonas gingivalis: possibleinvolvement of nonreciprocal recombination. Microbiol Immunol 48,185-196.

[0178] O'Brien-Simpson. N. M., Ede, N. J., Brown, L. E., Swan, J., andJackson, D. C. (1997). “Polymerisation of unprotected syntheticpeptides: a view towards a synthetic peptide vaccines.” J. Am. Chem.Soc. 117(6).

[0179] O'Sullivan, D., Arrhenius, T., Sidney, J., et al (1991). “On theinteraction of promiscuous antigenic peptides with different DR alleles.Indentification of common structural motifs.” J. Immunol 147(8):2663-2669.

[0180] Rose, K. (1994). “Facile synthesis of homogeneous artificialproteins.” J. Am. Chem. Soc. 116: 30-33.

[0181] Rose, J., Zeng, W., Regamey, P. O., Chernusheivich, I. V.,Standing, K. G., and Gaertner, H. F. (1996). “Natural peptides asbuilding blocks for the synthesis of large protein-like molecules withhydrazone and oxime linkages.” Bioconjugate Chem. 7(5): 552-556.

[0182] Shao, J., and Tam, J. P. (1995). J. Am. Chem. Soc. 117:3893-3899.

[0183] Slakeski. N., Cleal, S. M. & Reynolds, E. C. (1996).Characterization of a Porphyromonas gingivalis gene prtR that encodes anarginine-specific thiol proteinase and multiple adhesins. BiochemBiophys Res Comm 224, 605-610.

[0184] Spetzler, J. C. a. T., J. P. (1994). A general approach for thesynthesis of branched peptides for synthetic vaccines: Synthesis ofmultiple antigen peptides using unprotected segments. Innovation andPerspectives in Solid Phase Synthesis. R. Epton. Kingswinford,Mayflower: 293-300.

[0185] van Noort, J. M., and van der Drift, A. C. M. (1989). “Theselectivity of cathepsin D suggeste an involvement of the enzyme in thegeneration of T-cell epitopes.” J. Biol. Chem. 264(24): 14159-14164.

1 105 1 21 PRT Porphyromonas gingivalis 1 Phe Asn Gly Gly Ile Ser LeuAla Asn Tyr Thr Gly His Gly Ser Glu 1 5 10 15 Thr Ala Trp Gly Thr 20 222 PRT Porphyromonas gingivalis 2 Leu Asn Thr Gly Val Ser Phe Ala AsnTyr Thr Ala His Gly Ser Glu 1 5 10 15 Thr Ala Trp Ala Asp Pro 20 3 24PRT Porphyromonas gingivalis 3 Phe Asp Val Ala Cys Val Asn Gly Asp PheLeu Phe Ser Met Pro Cys 1 5 10 15 Phe Ala Glu Ala Leu Met Arg Ala 20 424 PRT Porphyromonas gingivalis 4 Ile Gly Asn Cys Cys Ile Thr Ala GlnPhe Asp Tyr Val Gln Pro Cys 1 5 10 15 Phe Gly Glu Val Ile Thr Arg Val 205 31 PRT Porphyromonas gingivalis 5 Gly Glu Pro Asn Pro Tyr Gln Pro ValSer Asn Leu Thr Ala Thr Thr 1 5 10 15 Gln Gly Gln Lys Val Thr Leu LysTrp Asp Ala Pro Ser Thr Lys 20 25 30 6 31 PRT Porphyromonas gingivalis 6Glu Gly Ser Asn Glu Phe Ala Pro Val Gln Asn Leu Thr Gly Ser Ala 1 5 1015 Val Gly Gln Lys Val Thr Leu Lys Trp Asp Ala Pro Asn Gly Thr 20 25 307 31 PRT Porphyromonas gingivalis 7 Val Asn Ser Thr Gln Phe Asn Pro ValLys Asn Leu Lys Ala Gln Pro 1 5 10 15 Asp Gly Gly Asp Val Val Leu LysTrp Glu Ala Pro Ser Ala Lys 20 25 30 8 31 PRT Porphyromonas gingivalis 8Gly Glu Pro Ser Pro Tyr Gln Pro Val Ser Asn Leu Thr Ala Thr Thr 1 5 1015 Gln Gly Gln Lys Val Thr Leu Lys Trp Glu Ala Pro Ser Ala Lys 20 25 309 31 PRT Porphyromonas gingivalis 9 Glu Gly Ser Asn Glu Phe Ala Pro ValGln Asn Leu Thr Gly Ser Ser 1 5 10 15 Val Gly Gln Lys Val Thr Leu LysTrp Asp Ala Pro Asn Gly Thr 20 25 30 10 33 PRT Porphyromonas gingivalis10 Val Asn Ser Thr Gln Phe Asn Pro Val Gln Asn Leu Thr Ala Glu Gln 1 510 15 Ala Pro Asn Ser Met Asp Ala Ile Leu Lys Trp Asn Ala Pro Ala Ser 2025 30 Lys 11 27 PRT Porphyromonas gingivalis 11 Gln Phe Asn Pro Val GlnAsn Leu Thr Gly Ser Ala Val Gly Gln Lys 1 5 10 15 Val Thr Leu Lys TrpAsp Ala Pro Asn Gly Thr 20 25 12 27 PRT Porphyromonas gingivalis 12 GlnPhe Asn Pro Val Gln Asn Leu Thr Gly Ser Ala Val Gly Gln Lys 1 5 10 15Val Thr Leu Lys Trp Asp Ala Pro Asn Gly Thr 20 25 13 27 PRTPorphyromonas gingivalis 13 Gln Phe Asn Pro Val Gln Asn Leu Thr Gly SerAla Val Gly Gln Lys 1 5 10 15 Val Thr Leu Lys Trp Asp Ala Pro Asn GlyThr 20 25 14 26 PRT Porphyromonas gingivalis 14 Phe Ala His Val Gln AsnLeu Thr Gly Ser Ala Val Gly Gln Lys Val 1 5 10 15 Thr Leu Lys Trp AspAla Pro Asn Gly Thr 20 25 15 26 PRT Porphyromonas gingivalis 15 Phe AlaPro Val Gln Asn Leu Gln Trp Ser Val Ser Gly Gln Thr Val 1 5 10 15 ThrLeu Thr Trp Gln Ala Pro Ala Ser Asp 20 25 16 29 PRT Porphyromonasgingivalis 16 Gln Phe Asn Pro Val Gln Asn Leu Thr Ala Glu Gln Ala ProAsn Ser 1 5 10 15 Met Asp Ala Ile Leu Lys Trp Asn Ala Pro Ala Ser Lys 2025 17 29 PRT Porphyromonas gingivalis 17 Asp Tyr Thr Tyr Thr Val Tyr ArgAsp Gly Thr Lys Ile Lys Glu Gly 1 5 10 15 Leu Thr Ala Thr Thr Phe GluGlu Asp Gly Val Ala Thr 20 25 18 29 PRT Porphyromonas gingivalis 18 AspTyr Thr Tyr Thr Val Tyr Arg Asp Gly Thr Lys Ile Lys Glu Gly 1 5 10 15Leu Thr Glu Thr Thr Phe Glu Glu Asp Gly Val Ala Thr 20 25 19 29 PRTPorphyromonas gingivalis 19 Ser Tyr Thr Tyr Thr Val Tyr Arg Asp Gly ThrLys Ile Lys Glu Gly 1 5 10 15 Leu Thr Glu Thr Thr Tyr Arg Asp Ala GlyMet Ser Ala 20 25 20 29 PRT Porphyromonas gingivalis 20 Ser Tyr Thr TyrThr Val Tyr Arg Asp Gly Thr Lys Ile Lys Glu Gly 1 5 10 15 Leu Thr AlaThr Thr Phe Glu Glu Asp Gly Val Ala Ala 20 25 21 29 PRT Porphyromonasgingivalis 21 Asp Tyr Thr Tyr Thr Val Tyr Arg Asp Gly Thr Lys Ile LysGlu Gly 1 5 10 15 Leu Thr Glu Thr Thr Phe Glu Glu Asp Gly Val Ala Thr 2025 22 29 PRT Porphyromonas gingivalis 22 Ser Tyr Thr Tyr Thr Ile Tyr ArgAsn Asn Thr Gln Ile Ala Ser Gly 1 5 10 15 Val Thr Glu Thr Thr Tyr ArgAsp Pro Asp Leu Ala Thr 20 25 23 29 PRT Porphyromonas gingivalis 23 AspTyr Thr Tyr Thr Val Tyr Arg Asp Gly Thr Lys Ile Lys Glu Gly 1 5 10 15Leu Thr Glu Thr Thr Phe Glu Glu Asp Gly Val Ala Thr 20 25 24 29 PRTPorphyromonas gingivalis 24 Asp Tyr Thr Tyr Thr Val Tyr Arg Asp Gly ThrLys Ile Lys Glu Gly 1 5 10 15 Leu Thr Glu Thr Thr Phe Glu Glu Asp GlyVal Ala Thr 20 25 25 29 PRT Porphyromonas gingivalis 25 Asp Tyr Thr TyrThr Val Tyr Arg Asp Gly Thr Lys Ile Lys Trp Gly 1 5 10 15 Leu Thr GluThr Thr Phe Glu Glu Asp Gly Val Ala Thr 20 25 26 29 PRT Porphyromonasgingivalis 26 Asp Tyr Thr Tyr Thr Val Tyr Arg Asp Gly Thr Lys Ile LysGlu Gly 1 5 10 15 Leu Thr Glu Thr Thr Phe Glu Glu Asp Gly Val Ala Thr 2025 27 29 PRT Porphyromonas gingivalis 27 Asp Tyr Thr Tyr Thr Val Tyr ArgAsp Asn Val Val Ile Ala Gln Asn 1 5 10 15 Leu Ala Ala Thr Thr Phe AsnGln Glu Asn Val Ala Pro 20 25 28 29 PRT Porphyromonas gingivalis 28 SerTyr Thr Tyr Thr Ile Tyr Arg Asn Asn Thr Gln Ile Ala Ser Gly 1 5 10 15Val Thr Glu Thr Thr Tyr Arg Asp Pro Asp Leu Ala Thr 20 25 29 26 PRTPorphyromonas gingivalis 29 Pro Asn Gly Thr Pro Asn Pro Asn Pro Asn ProAsn Pro Asn Pro Asn 1 5 10 15 Pro Gly Thr Thr Thr Leu Ser Glu Ser Phe 2025 30 23 PRT Porphyromonas gingivalis 30 Pro Asn Gly Thr Pro Asn Pro AsnPro Asn Pro Asn Pro Asn Pro Gly 1 5 10 15 Thr Thr Leu Ser Glu Ser Phe 2031 22 PRT Porphyromonas gingivalis 31 Pro Asn Gly Thr Pro Asn Pro AsnPro Asn Pro Asn Pro Gly Thr Thr 1 5 10 15 Thr Leu Ser Glu Ser Phe 20 3222 PRT Porphyromonas gingivalis 32 Pro Asn Gly Thr Pro Asn Pro Asn ProAsn Pro Asn Pro Gly Thr Thr 1 5 10 15 Thr Leu Ser Glu Ser Phe 20 33 22PRT Porphyromonas gingivalis 33 Pro Asn Gly Thr Pro Asn Pro Asn Pro AsnPro Asn Pro Gly Thr Thr 1 5 10 15 Thr Leu Ser Glu Ser Phe 20 34 18 PRTPorphyromonas gingivalis 34 Pro Asn Gly Thr Pro Asn Pro Asn Pro Gly ThrThr Thr Leu Ser Glu 1 5 10 15 Ser Phe 35 20 PRT Porphyromonas gingivalis35 Trp Ile Glu Arg Thr Val Asp Leu Pro Ala Gly Thr Lys Tyr Val Ala 1 510 15 Phe Arg His Tyr 20 36 20 PRT Porphyromonas gingivalis 36 Trp ArgGln Lys Thr Val Asp Leu Pro Ala Gly Thr Lys Tyr Val Ala 1 5 10 15 PheArg His Phe 20 37 20 PRT Porphyromonas gingivalis 37 Trp Ile Glu Arg ThrVal Asp Leu Pro Ala Gly Thr Lys Tyr Val Ala 1 5 10 15 Phe Arg His Tyr 2038 20 PRT Porphyromonas gingivalis 38 Trp Arg Gln Lys Thr Val Asp LeuPro Ala Gly Thr Lys Tyr Val Ala 1 5 10 15 Phe Arg His Phe 20 39 20 PRTPorphyromonas gingivalis 39 Trp Tyr Gln Lys Thr Val Gln Leu Pro Ala GlyThr Lys Tyr Val Ala 1 5 10 15 Phe Arg His Phe 20 40 20 PRT Porphyromonasgingivalis 40 Trp Ile Glu Arg Thr Val Asp Leu Pro Ala Gly Thr Lys TyrVal Ala 1 5 10 15 Phe Arg His Tyr 20 41 20 PRT Porphyromonas gingivalis41 Trp Ile Glu Arg Thr Val Asp Leu Pro Ala Gly Thr Lys Tyr Val Ala 1 510 15 Phe Arg His Tyr 20 42 20 PRT Porphyromonas gingivalis 42 Trp IleGlu Arg Thr Val Asp Leu Pro Ala Gly Thr Lys Tyr Val Ala 1 5 10 15 PheArg His Tyr 20 43 20 PRT Porphyromonas gingivalis 43 Trp Ile Glu Arg ThrVal Asp Leu Pro Ala Gly Thr Lys Tyr Val Ala 1 5 10 15 Phe Arg His Tyr 2044 20 PRT Porphyromonas gingivalis 44 Trp Tyr Gln Lys Thr Val Gln LeuPro Ala Gly Thr Lys Tyr Val Ala 1 5 10 15 Phe Arg His Phe 20 45 20 PRTPorphyromonas gingivalis 45 Trp Tyr Gln Lys Thr Val Gln Leu Pro Ala GlyThr Lys Tyr Val Ala 1 5 10 15 Phe Arg His Phe 20 46 20 PRT Porphyromonasgingivalis 46 Trp Tyr Gln Lys Thr Val Gln Leu Pro Ala Gly Thr Lys TyrVal Ala 1 5 10 15 Phe Arg His Phe 20 47 20 PRT Porphyromonas gingivalis47 Trp Tyr Gln Lys Thr Val Gln Leu Pro Ala Gly Thr Lys Tyr Val Ala 1 510 15 Phe Arg His Phe 20 48 20 PRT Porphyromonas gingivalis 48 Trp TyrGln Lys Thr Val Gln Leu Pro Ala Gly Thr Lys Tyr Val Ala 1 5 10 15 PheArg His Phe 20 49 21 PRT Porphyromonas gingivalis 49 Glu Arg Thr Ile AspLeu Ser Ala Tyr Ala Gly Gln Gln Val Tyr Leu 1 5 10 15 Ala Phe Arg HisPhe 20 50 16 PRT Porphyromonas gingivalis 50 Pro Ala Glu Trp Thr Thr IleAsp Ala Asp Gly Asp Gly Gln Gly Trp 1 5 10 15 51 16 PRT Porphyromonasgingivalis 51 Pro Ala Ser Trp Lys Thr Ile Asp Ala Asp Gly Asp Gly HisGly Trp 1 5 10 15 52 16 PRT Porphyromonas gingivalis 52 Pro Ala Glu TrpThr Thr Ile Asp Ala Asp Gly Asp Gly Gln Gly Trp 1 5 10 15 53 16 PRTPorphyromonas gingivalis 53 Pro Ala Ser Trp Lys Thr Ile Asp Ala Asp GlyAsp Gly His Gly Trp 1 5 10 15 54 16 PRT Porphyromonas gingivalis 54 ProAla Ser Trp Lys Thr Ile Asp Ala Asp Gly Asp Gly Asn Asn Trp 1 5 10 15 5516 PRT Porphyromonas gingivalis 55 Pro Ala Glu Trp Thr Thr Ile Asp AlaAsp Gly Asp Gly Gln Gly Trp 1 5 10 15 56 16 PRT Porphyromonas gingivalis56 Pro Ala Glu Trp Thr Thr Ile Asp Ala Asp Gly Asp Gly Gln Gly Trp 1 510 15 57 16 PRT Porphyromonas gingivalis 57 Pro Ala Glu Trp Thr Thr IleAsp Ala Asp Gly Asp Gly Gln Gly Trp 1 5 10 15 58 16 PRT Porphyromonasgingivalis 58 Pro Ala Glu Trp Thr Thr Ile Asp Ala Asp Gly Asp Gly GlnGly Trp 1 5 10 15 59 16 PRT Porphyromonas gingivalis 59 Pro Ala Ser TrpLys Thr Ile Asp Ala Asp Gly Asp Gly Asn Asn Trp 1 5 10 15 60 16 PRTPorphyromonas gingivalis 60 Pro Ala Ser Trp Lys Thr Ile Asp Ala Asp GlyAsp Gly Asn Asn Trp 1 5 10 15 61 16 PRT Porphyromonas gingivalis 61 ProAla Ser Trp Lys Thr Ile Asp Ala Asp Gly Asp Gly Asn Asn Trp 1 5 10 15 6216 PRT Porphyromonas gingivalis 62 Pro Ala Ser Trp Lys Thr Ile Asp AlaAsp Gly Asp Gly Asn Asn Trp 1 5 10 15 63 16 PRT Porphyromonas gingivalis63 Pro Ser Ser Trp Lys Thr Ile Asp Ala Asp Gly Asp Gly Asn Asn Trp 1 510 15 64 16 PRT Porphyromonas gingivalis 64 Pro Asn Gly Trp Thr Met IleAsp Ala Asp Gly Asp Gly His Asn Trp 1 5 10 15 65 20 PRT Porphyromonasgingivalis 65 Glu Gly Ser Asn Glu Phe Ala Pro Val Gln Asn Leu Thr GlySer Ala 1 5 10 15 Val Gly Gln Lys 20 66 20 PRT Porphyromonas gingivalis66 Gly Glu Pro Asn Pro Tyr Gln Pro Val Ser Asn Leu Thr Ala Thr Thr 1 510 15 Gln Gly Gln Lys 20 67 20 PRT Porphyromonas gingivalis 67 Glu GlySer Asn Glu Phe Ala Pro Val Gln Asn Leu Thr Gly Ser Ser 1 5 10 15 ValGly Gln Lys 20 68 20 PRT Porphyromonas gingivalis 68 Gly Glu Pro Ser ProTyr Gln Pro Val Ser Asn Leu Thr Ala Thr Thr 1 5 10 15 Gln Gly Gln Lys 2069 19 PRT Porphyromonas gingivalis 69 Asn Ser Thr Gln Phe Asn Pro ValGln Asn Leu Thr Ala Glu Gln Ala 1 5 10 15 Pro Asn Ser 70 20 PRTPorphyromonas gingivalis 70 Glu Gly Ser Asn Glu Phe Ala His Val Gln AsnLeu Thr Gly Ser Ala 1 5 10 15 Val Gly Gln Lys 20 71 19 PRT Porphyromonasgingivalis 71 Asp Pro Val Gln Phe Asn Pro Val Gln Asn Leu Thr Gly SerAla Val 1 5 10 15 Gly Gln Lys 72 19 PRT Porphyromonas gingivalis 72 AspPro Val Gln Phe Asn Pro Val Gln Asn Leu Thr Gly Ser Ala Val 1 5 10 15Gly Gln Lys 73 19 PRT Porphyromonas gingivalis 73 Asp Pro Val Gln PheAsn Pro Val Gln Asn Leu Thr Gly Ser Ala Val 1 5 10 15 Gly Gln Lys 74 20PRT Porphyromonas gingivalis 74 Glu Gly Gly Asn Glu Phe Ala Pro Val GlnAsn Leu Gln Trp Ser Val 1 5 10 15 Ser Gly Gln Thr 20 75 19 PRTPorphyromonas gingivalis 75 Asn Pro Thr Gln Phe Asn Pro Val Gln Asn LeuThr Ala Glu Gln Ala 1 5 10 15 Pro Asn Ser 76 25 PRT Porphyromonasgingivalis 76 Gly Asn His Glu Tyr Cys Val Glu Val Lys Tyr Thr Ala GlyVal Ser 1 5 10 15 Pro Lys Val Cys Lys Asp Val Thr Val 20 25 77 25 PRTPorphyromonas gingivalis 77 Gly Asn His Glu Tyr Cys Val Glu Val Lys TyrThr Ala Gly Val Ser 1 5 10 15 Pro Lys Lys Cys Val Asn Val Thr Val 20 2578 21 PRT Porphyromonas gingivalis 78 Ser His Glu Tyr Cys Val Glu ValLys Tyr Thr Ala Gly Val Ser Pro 1 5 10 15 Lys Val Cys Val Asp 20 79 25PRT Porphyromonas gingivalis 79 Gly Asn His Glu Tyr Cys Val Glu Val LysTyr Thr Ala Gly Val Ser 1 5 10 15 Pro Lys Val Cys Lys Asp Val Thr Val 2025 80 25 PRT Porphyromonas gingivalis 80 Gly Asn His Glu Tyr Cys Val GluVal Lys Tyr Thr Ala Gly Val Ser 1 5 10 15 Pro Lys Lys Cys Val Asn ValThr Val 20 25 81 25 PRT Porphyromonas gingivalis 81 Gly Asn His Glu TyrCys Val Glu Val Lys Tyr Thr Ala Gly Val Ser 1 5 10 15 Pro Lys Val CysVal Asn Val Thr Ile 20 25 82 25 PRT Porphyromonas gingivalis 82 Gly GlnTyr Asn Tyr Cys Val Glu Val Lys Tyr Thr Ala Gly Val Ser 1 5 10 15 ProLys Val Cys Lys Asp Val Thr Val 20 25 83 25 PRT Porphyromonas gingivalis83 Gly Asn His Glu Tyr Cys Val Glu Val Lys Tyr Thr Ala Gly Val Ser 1 510 15 Pro Glu Val Cys Val Asn Val Thr Val 20 25 84 25 PRT Porphyromonasgingivalis 84 Gly Asn His Glu Tyr Cys Val Glu Val Lys Tyr Thr Ala GlyVal Ser 1 5 10 15 Pro Glu Val Cys Val Asn Val Thr Val 20 25 85 25 PRTPorphyromonas gingivalis 85 Gly Asn His Glu Tyr Cys Val Glu Val Lys TyrThr Ala Gly Val Ser 1 5 10 15 Pro Glu Val Cys Val Asn Val Thr Val 20 2586 27 PRT Porphyromonas gingivalis 86 Pro Tyr Gln Pro Val Ser Asn LeuThr Ala Thr Thr Gln Gly Gln Lys 1 5 10 15 Val Thr Leu Lys Trp Asp AlaPro Ser Thr Lys 20 25 87 16 DNA Porphyromonas gingivalis 87 cggcttccgtaaagtc 16 88 24 DNA Porphyromonas gingivalis 88 tggctacgat gacgatcatacgac 24 89 26 PRT Porphyromonas gingivalis 89 Tyr Thr Pro Val Glu GluLys Glu Asn Gly Arg Met Ile Val Ile Val 1 5 10 15 Pro Lys Lys Tyr GluGlu Asp Ile Glu Asp 20 25 90 6 PRT Porphyromonas gingivalis VARIANT 3Xaa = Any Amino Acid 90 Pro Val Xaa Asn Leu Thr 1 5 91 6 PRTPorphyromonas gingivalis VARIANT 4 Xaa = Any Amino Acid 91 Leu Lys TrpXaa Ala Pro 1 5 92 11 PRT Porphyromonas gingivalis 92 Thr Ala Thr ThrPhe Glu Glu Asp Gly Val Ala 1 5 10 93 10 PRT Porphyromonas gingivalis 93Trp Lys Thr Ile Asp Ala Asp Gly Asp Gly 1 5 10 94 10 PRT Porphyromonasgingivalis 94 Val Tyr Arg Asp Gly Thr Lys Ile Lys Glu 1 5 10 95 15 PRTPorphyromonas gingivalis 95 Trp Glu Ile Arg Thr Val Asp Leu Pro Ala GlyThr Lys Tyr Val 1 5 10 15 96 12 PRT Porphyromonas gingivalis 96 Glu PheAla Pro Val Gln Asn Leu Thr Gly Ser Ala 1 5 10 97 32 PRT Porphyromonasgingivalis 97 Ala Asn Glu Ala Lys Val Val Leu Ala Ala Asp Asn Val TrpGly Asp 1 5 10 15 Asn Thr Gly Tyr Gln Phe Leu Leu Asp Ala Asp His AsnThr Phe Gly 20 25 30 98 33 PRT Porphyromonas gingivalis 98 Val Thr LeuLys Trp Asp Ala Pro Asn Gly Thr Pro Asn Pro Asn Pro 1 5 10 15 Asn ProAsn Pro Asn Pro Asn Pro Gly Thr Thr Thr Leu Ser Glu Ser 20 25 30 Phe 99507 PRT Porphyromonas gingivalis 99 Tyr Thr Pro Val Glu Glu Lys Glu AsnGly Arg Met Ile Val Ile Val 1 5 10 15 Pro Lys Lys Tyr Glu Glu Asp IleGlu Asp Phe Val Asp Trp Lys Asn 20 25 30 Gln Arg Gly Leu Arg Thr Glu ValLys Val Ala Glu Asp Ile Ala Ser 35 40 45 Pro Val Thr Ala Asn Ala Ile GlnGln Phe Val Lys Gln Glu Tyr Glu 50 55 60 Lys Glu Gly Asn Asp Leu Thr TyrVal Leu Leu Val Gly Asp His Lys 65 70 75 80 Asp Ile Pro Ala Lys Ile ThrPro Gly Ile Lys Ser Asp Gln Val Tyr 85 90 95 Gly Gln Ile Val Gly Asn AspHis Tyr Asn Glu Val Phe Ile Gly Arg 100 105 110 Phe Ser Cys Glu Ser LysGlu Asp Leu Lys Thr Gln Ile Asp Arg Thr 115 120 125 Ile His Tyr Glu ArgAsn Ile Thr Thr Glu Asp Lys Trp Leu Gly Gln 130 135 140 Ala Leu Cys IleAla Ser Ala Glu Gly Gly Pro Ser Ala Asp Asn Gly 145 150 155 160 Glu SerAsp Ile Gln His Glu Asn Ile Ile Ala Asn Leu Leu Thr Gln 165 170 175 TyrGly Tyr Thr Lys Ile Ile Lys Cys Tyr Asp Pro Gly Val Thr Pro 180 185 190Lys Asn Ile Ile Asp Ala Phe Asn Gly Gly Ile Ser Leu Ala Asn Tyr 195 200205 Thr Gly His Gly Ser Glu Thr Ala Trp Gly Thr Ser His Phe Gly Thr 210215 220 Thr His Val Lys Gln Leu Thr Asn Ser Asn Gln Leu Pro Phe Ile Phe225 230 235 240 Asp Val Ala Cys Val Asn Gly Asp Phe Leu Tyr Asn Val ProCys Phe 245 250 255 Ala Glu Ala Leu Met Arg Ala Gln Lys Asp Gly Lys ProThr Gly Thr 260 265 270 Val Ala Ile Ile Ala Ser Thr Ile Asn Gln Ser TrpAla Ser Pro Met 275 280 285 Arg Gly Gln Asp Glu Met Asn Glu Ile Leu CysGlu Lys His Pro Asn 290 295 300 Asn Ile Lys Arg Thr Phe Gly Gly Val ThrMet Asn Gly Met Phe Ala 305 310 315 320 Met Val Glu Lys Tyr Lys Lys AspGly Glu Lys Met Leu Asp Thr Trp 325 330 335 Thr Val Phe Gly Asp Pro SerLeu Leu Val Arg Thr Leu Val Pro Thr 340 345 350 Lys Met Gln Val Thr AlaPro Ala Asn Ile Ser Ala Ser Ala Gln Thr 355 360 365 Phe Glu Val Ala CysAsp Tyr Asn Gly Ala Ile Ala Thr Leu Ser Asp 370 375 380 Asp Gly Asp MetVal Gly Thr Ala Ile Val Lys Asp Gly Lys Ala Ile 385 390 395 400 Ile LysLeu Asn Glu Ser Ile Ala Asp Glu Thr Asn Leu Thr Leu Thr 405 410 415 ValVal Gly Tyr Asn Lys Val Thr Val Ile Lys Asp Val Lys Val Glu 420 425 430Gly Thr Ser Ile Ala Asp Val Ala Asn Asp Lys Pro Tyr Thr Val Ala 435 440445 Val Ser Gly Lys Thr Ile Thr Val Glu Ser Pro Ala Ala Gly Leu Thr 450455 460 Ile Phe Asp Met Asn Gly Arg Arg Val Ala Thr Ala Lys Asn Arg Met465 470 475 480 Val Phe Glu Ala Gln Asn Gly Val Tyr Ala Val Arg Ile AlaThr Glu 485 490 495 Gly Lys Thr Tyr Thr Glu Lys Val Ile Val Lys 500 505100 491 PRT Porphyromonas gingivalis 100 Tyr Thr Pro Val Glu Glu Lys GlnAsn Gly Arg Met Ile Val Ile Val 1 5 10 15 Ala Lys Lys Tyr Glu Gly AspIle Lys Asp Phe Val Asp Trp Lys Asn 20 25 30 Gln Arg Gly Leu Arg Thr GluVal Lys Val Ala Glu Asp Ile Ala Ser 35 40 45 Pro Val Thr Ala Asn Ala IleGln Gln Phe Val Lys Gln Glu Tyr Glu 50 55 60 Lys Glu Gly Asn Asp Leu ThrTyr Val Leu Leu Ile Gly Asp His Lys 65 70 75 80 Asp Ile Pro Ala Lys IleThr Pro Gly Ile Lys Ser Asp Gln Val Tyr 85 90 95 Gly Gln Ile Val Gly AsnAsp His Tyr Asn Glu Val Phe Ile Gly Arg 100 105 110 Phe Ser Cys Glu SerLys Glu Asp Leu Lys Thr Gln Ile Asp Arg Thr 115 120 125 Ile His Tyr GluArg Asn Ile Thr Thr Glu Asp Lys Trp Leu Gly Gln 130 135 140 Ala Leu CysIle Ala Ser Ala Glu Gly Gly Pro Ser Ala Asp Asn Gly 145 150 155 160 GluSer Asp Ile Gln His Glu Asn Val Ile Ala Asn Leu Leu Thr Gln 165 170 175Tyr Gly Tyr Thr Lys Ile Ile Lys Cys Tyr Asp Pro Gly Val Thr Pro 180 185190 Lys Asn Ile Ile Asp Ala Phe Asn Gly Gly Ile Ser Leu Ala Asn Tyr 195200 205 Thr Gly His Gly Ser Glu Thr Ala Trp Gly Thr Ser His Phe Gly Thr210 215 220 Thr His Val Lys Gln Leu Thr Asn Ser Asn Gln Leu Pro Phe IlePhe 225 230 235 240 Asp Val Ala Cys Val Asn Gly Asp Phe Leu Phe Ser MetPro Cys Phe 245 250 255 Ala Glu Ala Leu Met Arg Ala Gln Lys Asp Gly LysPro Thr Gly Val 260 265 270 Ala Ile Ile Ala Ser Thr Ile Asn Gln Ser TrpAla Ser Pro Met Arg 275 280 285 Gly Gln Asp Glu Met Asn Glu Ile Leu CysGlu Lys His Pro Asn Asn 290 295 300 Ile Lys Arg Thr Phe Gly Gly Val ThrMet Asn Gly Met Phe Ala Met 305 310 315 320 Val Glu Lys Tyr Lys Lys AspGly Glu Lys Met Leu Asp Thr Trp Thr 325 330 335 Val Phe Gly Asp Pro SerLeu Leu Val Arg Thr Leu Val Pro Thr Lys 340 345 350 Met Gln Val Thr AlaPro Ala Gln Ile Asn Leu Thr Asp Ala Ser Val 355 360 365 Asn Val Ser CysAsp Tyr Asn Gly Ala Ile Ala Thr Ile Ser Ala Asn 370 375 380 Gly Lys MetPhe Gly Ser Ala Val Val Glu Asn Gly Thr Ala Thr Ile 385 390 395 400 AsnLeu Thr Gly Leu Thr Asn Glu Ser Thr Leu Thr Leu Thr Val Val 405 410 415Gly Tyr Asn Lys Glu Thr Val Ile Lys Thr Ile Asn Thr Asn Gly Glu 420 425430 Pro Asn Pro Tyr Gln Pro Val Ser Asn Leu Thr Ala Thr Thr Gln Gly 435440 445 Gln Lys Val Thr Leu Lys Trp Asp Ala Pro Ser Thr Lys Thr Asn Ala450 455 460 Thr Thr Asn Thr Ala Arg Ser Val Asp Gly Ile Arg Glu Leu ValLeu 465 470 475 480 Leu Ser Val Ser Asp Ala Pro Glu Leu Leu Arg 485 490101 509 PRT Porphyromonas gingivalis 101 Asp Val Tyr Thr Asp His Gly AspLeu Tyr Asn Thr Pro Val Arg Met 1 5 10 15 Leu Val Val Ala Gly Ala LysPhe Lys Glu Ala Leu Lys Pro Trp Leu 20 25 30 Thr Trp Lys Ala Gln Lys GlyPhe Tyr Leu Asp Val His Tyr Thr Asp 35 40 45 Glu Ala Glu Val Gly Thr ThrAsn Ala Ser Ile Lys Ala Phe Ile His 50 55 60 Lys Lys Tyr Asn Asp Gly LeuAla Ala Ser Ala Ala Pro Val Phe Leu 65 70 75 80 Ala Leu Val Gly Asp ThrAsp Val Ile Ser Gly Glu Lys Gly Lys Lys 85 90 95 Thr Lys Lys Val Thr AspLeu Tyr Tyr Ser Ala Val Asp Gly Asp Tyr 100 105 110 Phe Pro Glu Met TyrThr Phe Arg Met Ser Ala Ser Ser Pro Glu Glu 115 120 125 Leu Thr Asn IleIle Asp Lys Val Leu Met Tyr Glu Lys Ala Thr Met 130 135 140 Pro Asp LysSer Tyr Leu Glu Lys Val Leu Leu Ile Ala Gly Ala Asp 145 150 155 160 TyrSer Trp Asn Ser Gln Val Gly Gln Pro Thr Ile Lys Tyr Gly Met 165 170 175Gln Tyr Tyr Tyr Asn Gln Glu His Gly Tyr Thr Asp Val Tyr Asn Tyr 180 185190 Leu Lys Ala Pro Tyr Thr Gly Cys Tyr Ser His Leu Asn Thr Gly Val 195200 205 Ser Phe Ala Asn Tyr Thr Ala His Gly Ser Glu Thr Ala Trp Ala Asp210 215 220 Pro Leu Leu Thr Thr Ser Gln Leu Lys Ala Leu Thr Asn Lys AspLys 225 230 235 240 Tyr Phe Leu Ala Ile Gly Asn Cys Cys Ile Thr Ala GlnPhe Asp Tyr 245 250 255 Val Gln Pro Cys Phe Gly Glu Val Ile Thr Arg ValLys Glu Lys Gly 260 265 270 Ala Tyr Ala Tyr Ile Gly Ser Ser Pro Asn SerTyr Trp Gly Glu Asp 275 280 285 Tyr Tyr Trp Ser Val Gly Ala Asn Ala ValPhe Gly Val Gln Pro Thr 290 295 300 Phe Glu Gly Thr Ser Met Gly Ser TyrAsp Ala Thr Phe Leu Glu Asp 305 310 315 320 Ser Tyr Asn Thr Val Asn SerIle Met Trp Ala Gly Asn Leu Ala Ala 325 330 335 Thr His Ala Gly Asn IleGly Asn Ile Thr His Ile Gly Ala His Tyr 340 345 350 Tyr Trp Glu Ala TyrHis Val Leu Gly Asp Gly Ser Val Met Pro Tyr 355 360 365 Arg Ala Met ProLys Thr Asn Thr Tyr Thr Leu Pro Ala Ser Leu Pro 370 375 380 Gln Asn GlnAla Ser Tyr Ser Ile Gln Ala Ser Ala Gly Ser Tyr Val 385 390 395 400 AlaIle Ser Lys Asp Gly Val Leu Tyr Gly Thr Gly Val Ala Asn Ala 405 410 415Ser Gly Val Ala Thr Val Ser Met Thr Lys Gln Ile Thr Glu Asn Gly 420 425430 Asn Tyr Asp Val Val Ile Thr Arg Ser Asn Tyr Leu Pro Val Ile Lys 435440 445 Gln Ile Gln Val Gly Glu Pro Ser Pro Tyr Gln Pro Val Ser Asn Leu450 455 460 Thr Ala Thr Thr Gln Gly Gln Lys Val Thr Leu Lys Trp Glu AlaPro 465 470 475 480 Ser Ala Lys Lys Ala Glu Gly Ser Arg Glu Val Lys ArgIle Gly Asp 485 490 495 Gly Leu Phe Val Thr Ile Glu Pro Ala Asn Asp ValArg 500 505 102 4 PRT Porphyromonas gingivalis 102 Thr Leu Cys Lys 1 10311 PRT Porphyromonas gingivalis 103 Thr Ala Thr Thr Phe Glu Glu Asp GlyVal Ala 1 5 10 104 10 PRT Porphyromonas gingivalis 104 Trp Lys Thr IleAsp Ala Asp Gly Asp Gly 1 5 10 105 736 PRT Porphyromonas gingivalis 105Met Lys Lys Asn Phe Ser Arg Ile Val Ser Ile Val Ala Phe Ser Ser 1 5 1015 Leu Leu Gly Gly Met Ala Phe Ala Gln Pro Ala Glu Arg Gly Arg Asn 20 2530 Pro Gln Val Arg Leu Leu Ser Ala Glu Gln Ser Met Ser Lys Val Gln 35 4045 Phe Arg Met Asp Asn Leu Gln Phe Thr Gly Val Gln Thr Ser Lys Gly 50 5560 Val Ala Gln Val Pro Thr Phe Thr Glu Gly Val Asn Ile Ser Glu Lys 65 7075 80 Gly Thr Pro Ile Leu Pro Ile Leu Ser Arg Ser Leu Ala Val Ser Glu 8590 95 Thr Arg Ala Met Lys Val Glu Val Val Ser Ser Lys Phe Ile Glu Lys100 105 110 Lys Asp Val Leu Ile Ala Pro Ser Lys Gly Val Ile Ser Arg AlaGlu 115 120 125 Asn Pro Asp Gln Ile Pro Tyr Val Tyr Gly Gln Ser Tyr AsnGlu Asp 130 135 140 Lys Phe Phe Pro Gly Glu Ile Ala Thr Leu Ser Asp ProPhe Ile Leu 145 150 155 160 Arg Asp Val Arg Gly Gln Val Val Asn Phe AlaPro Leu Gln Tyr Asn 165 170 175 Pro Val Thr Lys Thr Leu Arg Ile Tyr ThrGlu Ile Val Val Ala Val 180 185 190 Ser Glu Thr Ala Glu Ala Gly Gln AsnThr Ile Ser Leu Val Lys Asn 195 200 205 Ser Thr Phe Thr Gly Phe Glu AspIle Tyr Lys Ser Val Phe Met Asn 210 215 220 Tyr Glu Ala Thr Arg Tyr ThrPro Val Glu Glu Lys Glu Asn Gly Arg 225 230 235 240 Met Ile Val Ile ValPro Lys Lys Tyr Glu Glu Asp Ile Glu Asp Phe 245 250 255 Val Asp Trp LysAsn Gln Arg Gly Leu Arg Thr Glu Val Lys Val Ala 260 265 270 Glu Asp IleAla Ser Pro Val Thr Ala Asn Ala Ile Gln Gln Phe Val 275 280 285 Lys GlnGlu Tyr Glu Lys Glu Gly Asn Asp Leu Thr Tyr Val Leu Leu 290 295 300 ValGly Asp His Lys Asp Ile Pro Ala Lys Ile Thr Pro Gly Ile Lys 305 310 315320 Ser Asp Gln Val Tyr Gly Gln Ile Val Gly Asn Asp His Tyr Asn Glu 325330 335 Val Phe Ile Gly Arg Phe Ser Cys Glu Ser Lys Glu Asp Leu Lys Thr340 345 350 Gln Ile Asp Arg Thr Ile His Tyr Glu Arg Asn Ile Thr Thr GluAsp 355 360 365 Lys Trp Leu Gly Gln Ala Leu Cys Ile Ala Ser Ala Glu GlyGly Pro 370 375 380 Ser Ala Asp Asn Gly Glu Ser Asp Ile Gln His Glu AsnIle Ile Ala 385 390 395 400 Asn Leu Leu Thr Gln Tyr Gly Tyr Thr Lys IleIle Lys Cys Tyr Asp 405 410 415 Pro Gly Val Thr Pro Lys Asn Ile Ile AspAla Phe Asn Gly Gly Ile 420 425 430 Ser Leu Ala Asn Tyr Thr Gly His GlySer Glu Thr Ala Trp Gly Thr 435 440 445 Ser His Phe Gly Thr Thr His ValLys Gln Leu Thr Asn Ser Asn Gln 450 455 460 Leu Pro Phe Ile Phe Asp ValAla Cys Val Asn Gly Asp Phe Leu Tyr 465 470 475 480 Asn Val Pro Cys PheAla Glu Ala Leu Met Arg Ala Gln Lys Asp Gly 485 490 495 Lys Pro Thr GlyThr Val Ala Ile Ile Ala Ser Thr Ile Asn Gln Ser 500 505 510 Trp Ala SerPro Met Arg Gly Gln Asp Glu Met Asn Glu Ile Leu Cys 515 520 525 Glu LysHis Pro Asn Asn Ile Lys Arg Thr Phe Gly Gly Val Thr Met 530 535 540 AsnGly Met Phe Ala Met Val Glu Lys Tyr Lys Lys Asp Gly Glu Lys 545 550 555560 Met Leu Asp Thr Trp Thr Val Phe Gly Asp Pro Ser Leu Leu Val Arg 565570 575 Thr Leu Val Pro Thr Lys Met Gln Val Thr Ala Pro Ala Asn Ile Ser580 585 590 Ala Ser Ala Gln Thr Phe Glu Val Ala Cys Asp Tyr Asn Gly AlaIle 595 600 605 Ala Thr Leu Ser Asp Asp Gly Asp Met Val Gly Thr Ala IleVal Lys 610 615 620 Asp Gly Lys Ala Ile Ile Lys Leu Asn Glu Ser Ile AlaAsp Glu Thr 625 630 635 640 Asn Leu Thr Leu Thr Val Val Gly Tyr Asn LysVal Thr Val Ile Lys 645 650 655 Asp Val Lys Val Glu Gly Thr Ser Ile AlaAsp Val Ala Asn Asp Lys 660 665 670 Pro Tyr Thr Val Ala Val Ser Gly LysThr Ile Thr Val Glu Ser Pro 675 680 685 Ala Ala Gly Leu Thr Ile Phe AspMet Asn Gly Arg Arg Val Ala Thr 690 695 700 Ala Lys Asn Arg Met Val PheGlu Ala Gln Asn Gly Val Tyr Ala Val 705 710 715 720 Arg Ile Ala Thr GluGly Lys Thr Tyr Thr Glu Lys Val Ile Val Lys 725 730 735

1. A composition for use in raising an immune response againstPorphyromonas gingivalis, the composition including a suitable adjuvantand/or acceptable carrier or excipient and at least one peptide selectedfrom the group consisting of:— FNGGISLANYTGHGSETAWGT;LNTGVSFANYTAHGSETAWADP; FDVACVNGDFLFSMPCFAEALMRA;IGNCCITAQFDYVQPCFGEVITRV; GEPNPYQPVSNLTATTQGQKVTLKWDAPSTK;EGSNEFAPVQNLTGSAVGQKVTLKWDAPNGT; VNSTQFNPVKNLKAQPDGGDVVLKWEAPSAK;GEPSPYQPVSNLTATTQGQKVTLKWEAPSAK; EGSNEFAPVQNLTGSSVGQKVTLKWDAPNGT;VNSTQFNPVQNLTAEQAPNSMDAILKWNAPASK; QFNPVQNLTGSAVGQKVTLKWDAPNGT;FAHVQNLTGSAVGQKVTLKWDAPNGT; FAPVQNLQWSVSGQTVTLTWQAPASD;QFNPVQNLTAEQAPNSMDAILKWNAPASK; DYTYTVYRDGTKIKEGLTATTFEEDGVAT;DYTYTVYRDGTKIKEGLTETTFEEDGVAT; SYTYTVYRDGTKIKEGLTETTYRDAGMSA;SYTYTVYRDGTKIKEGLTATTFEEDGVAA; DYTYTVYRDGTKIKEGLTETTFEEDGVAT;SYTYTIYRNNTQIASGVTETTYRDPDLAT; DYTYTVYRDNVVIAQNLAATTFNQENVAP;SYTYTIYRNNTQIASGVTETTYRDPDLAT; PNGTPNPNPNPNPNPNPGTTTLSESF;PNGTPNPNPNPNPNPGTTLSESF; PNGTPNPNPNPNPGTTTLSESF; PNGTPNPNPGTTTLSESF;WIERTVDLPAGTKYVAFRHY; WRQKTVDLPAGTKYVAFRHF; WYQKTVQLPAGTKYVAFRHF;ERTIDLSAYAGQQVYLAFRHF; PAEWTTIDADGDGQGW; PASWKTIDADGDGHGW;PASWKTIDADGDGNNW; PSSWKTIDADGDGNNW; PNGWTMIDADGDGHNW;EGSNEFAPVQNLTGSAVGQK; GEPNPYQPVSNLTATTQGQK; EGSNEFAPVQNLTGSSVGQK;GEPSPYQPVSNLTATTQGQK; NSTQFNPVQNLTAEQAPNS; EGSNEFAHVQNLTGSAVGQK;DPVQFNPVQNLTGSAVGQK; EGGNEFAPVQNLQWSVSGQT; NPTQFNPVQNLTAEQAPNS;GNHEYCVEVKYTAGVSPKVCKDVTV; GNHEYCVEVKYTAGVSPKKCVNVTV;SHEYCVEVKYTAGVSPKVCVD; GNHEYCVEVKYTAGVSPKKCVNVTV;GNHEYCVEVKYTAGVSPKVCVNVTI; GQYNYCVEVKYTAGVSPKVCKDVTV; andGNHEYCVEVKYTAGVSPEVCVNVTV.


2. A composition as claimed in claim 1 in which the composition includesat least one peptide selected from the group consisting of:—FNGGISLANYTGHGSETAWGT; LNTGVSFANYTAhGSETAWADP;PYQPVSNLTATTQGQKVTLKWDAPSTK; SYTYTVYRDGTKIKEGLTATTFEEDGVAA;VTLKWDAPNGTPNPNPNPNPNPNPGTTTLSESF; WIERTVDLPAGTKYVAFRHY;PAEWTTIDADGDGQGW; and EGSNEFAPVQNLTGSAVGQK.


3. A composition as claimed in claim 1 or claim 2 in which thecomposition includes more than one peptide.
 4. A composition as claimedin claim 3 in which the composition includes one or more multimers ofdifferent peptides.
 5. A peptide selected from the group consisting of:—FNGGISLANYTGHGSETAWGT; LNTGVSFANYTAHGSETAWADP; FDVACVNGDFLFSMPCFAEALMRA;IGNCCITAQFDYVQPCFGEVITRV; GEPNPYQPVSNLTATTQGQKVTLKWDAPSTK;EGSNEFAPVQNLTGSAVGQKVTLKWDAPNGT; VNSTQFNPVKNLKAQPDGGDVVLKWEAPSAK;GEPSPYQPVSNLTATTQGQKVTLKWEAPSAK; EGSNEFAPVQNLTGSSVGQKVTLKWDAPNGT;VNSTQFNPVQNLTAEQAPNSMDAILKWNAPASK; QFNPVQNLTGSAVGQKVTLKWDAPNGT;FAHVQNLTGSAVGQKVTLKWDAPNGT; FAPVQNLQWSVSGQTVTLTWQAPASD;QFNPVQNLTAEQAPNSMDAILKWNAPASK; DYTYTVYRDGTKIKEGLTATTFEEDGVAT;DYTYTVYRDGTKIKEGLTETTFEEDGVAT; SYTYTVYRDGTKIKEGLTETTYRDAGMSA;SYTYTVYRDGTKIKEGLTATTFEEDGVAA; DYTYTVYRDGTKIKEGLTETTFEEDGVAT;SYTYTIYRNNTQIASGVTETTYRDPDLAT; DYTYTVYRDNVVIAQNLAATTFNQENVAP;SYTYTIYRNNTQIASGVTETTYRDPDLAT; PNGTPNPNPNPNPNPNPGTTTLSESF;PNGTPNPNPNPNPNPGTTLSESF; PNGTPNPNPNPNPGTTTLSESF; PNGTPNPNPGTTTLSESF;WIERTVDLPAGTKYVAFRHY; WRQKTVDLPAGTKYVAFRHF; WYQKTVQLPAGTKYVAFRHF;ERTIDLSAYAGQQVYLAFRHF; PAEWTTIDADGDGQGW; PASWKTIDADGDGHGW;PASWKTIDADGDGNNW; PSSWKTIDADGDGNNW; PNGWTMIDADGDGHNW;EGSNEFAPVQNLTGSAVGQK; GEPNPYQPVSNLTATTQGQK; EGSNEFAPVQNLTGSSVGQK;GEPSPYQPVSNLTATTQGQK; NSTQFNPVQNLTAEQAPNS; EGSNEFAHVQNLTGSAVGQK;DPVQFNPVQNLTGSAVGQK; EGGNEFAPVQNLQWSVSGQT; NPTQFNPVQNLTAEQAPNS;GNHEYCVEVKYTAGVSPKVCKDVTV; GNHEYCVEVKYTAGVSPKKCVNVTV;SHEYCVEVKYTAGVSPKVCVD; GNHEYCVEVKYTAGVSPKKCVNVTV;GNHEYCVEVKYTAGVSPKVCVNVTI; GQYNYCVEVKYTAGVSPKVCKDVTV; andGNHEYCVEVKYTAGVSPEVCVNVTV.


6. A peptide as claimed in claim 5 selected from the group consistingof:— FNGGISLANYTGHGSETAWGT; LNTGVSFANYTAHGSETAWADP;PYQPVSNLTATTQGQKVTLKWDAPSTK; SYTYTVYRDGTKIKEGLTATTFEEDGVAA;VTLKWDAPNGTPNPNPNPNPNPNPGTTTLSESF; WIERTVDLPAGTKYVAFRHY;PAEWTTIDADGDGQGW; and EGSNEFAPVQNLTGSAVGQK.


7. An antibody preparation comprising antibodies specifically directedagainst a peptide as claimed in claim 5 or claim
 6. 8. An antibodypreparation as claimed in claim 7 in which the antibodies are polyclonalantibodies.
 9. An antibody preparation as claimed in claim 7 in whichthe antibodies are monoclonal antibodies.
 10. A method of treating asubject suffering from Porphyromonas gingivalis infection, the methodcomprising administering to the subject an effective amount of acomposition as claimed in any one of claims 1 to 4 or a peptide asclaimed in claim 5 or claim
 6. 11. A method according to claim 10 inwhich the composition or peptide is administered as a mouth wash or as adentifrice.
 12. A method of treating a subject suffering fromPorphyromonas gingivalis infection, the method comprising administeringto the subject an effective amount of an antibody preparation as claimedin any one of claims 7 to
 9. 13. A method as claimed in claim 12 inwhich the antibody preparation is administered as a mouth wash or as adentifrice.
 14. A method of reducing the prospect of P. gingivalisinfection in an individual and/or severity of disease, the methodcomprising administering to the individual an amount of a composition asclaimed in any one of claims 1 to 4 effective to induce an immuneresponse in the individual directed against P. gingivalis.